Nucleobase editors and uses thereof

ABSTRACT

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing proteins or protein domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing proteins or domains, are provided.

RELATED APPLICATIONS

This application is a continuation of and claims priority under 35 U.S.C. § 120 to U.S. application, U.S. Ser. No. 15/960,171, filed Apr. 23, 2018, which is a continuation of and claims priority under 35 U.S.C. § 120 to international PCT Application, PCT/US2016/058344, filed Oct. 22, 2016, and is a continuation of and claims priority under 35 U.S.C. § 120 to U.S. application, U.S. Ser. No. 15/331,852, filed Oct. 22, 2016, which claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent applications, U.S. Ser. No. 62/245,828 filed Oct. 23, 2015, U.S. Ser. No. 62/279,346 filed Jan. 15, 2016, U.S. Ser. No. 62/311,763 filed Mar. 22, 2016, U.S. Ser. No. 62/322,178 filed Apr. 13, 2016, U.S. Ser. No. 62/357,352 filed Jun. 30, 2016, U.S. Ser. No. 62/370,700 filed Aug. 3, 2016, U.S. Ser. No. 62/398,490 filed Sep. 22, 2016, U.S. Ser. No. 62/408,686 filed Oct. 14, 2016, and U.S. Ser. No. 62/357,332 filed Jun. 30, 2016; each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under grant number EB022376 (formerly GM065400) awarded by the National Institutes of Health. The Government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 9, 2021, is named H082470213US10-SEQ-EPG and is 4,463,178 bytes in size.

BACKGROUND OF THE INVENTION

Targeted editing of nucleic acid sequences, for example, the targeted cleavage or the targeted introduction of a specific modification into genomic DNA, is a highly promising approach for the study of gene function and also has the potential to provide new therapies for human genetic diseases.¹ An ideal nucleic acid editing technology possesses three characteristics: (1) high efficiency of installing the desired modification; (2) minimal off-target activity; and (3) the ability to be programmed to edit precisely any site in a given nucleic acid, e.g., any site within the human genome.² Current genome engineering tools, including engineered zinc finger nucleases (ZFNs),³ transcription activator like effector nucleases (TALENs),⁴ and most recently, the RNA-guided DNA endonuclease Cas9,⁵ effect sequence-specific DNA cleavage in a genome. This programmable cleavage can result in mutation of the DNA at the cleavage site via non-homologous end joining (NHEJ) or replacement of the DNA surrounding the cleavage site via homology-directed repair (HDR).^(6,7)

One drawback to the current technologies is that both NHEJ and HDR are stochastic processes that typically result in modest gene editing efficiencies as well as unwanted gene alterations that can compete with the desired alteration.⁸ Since many genetic diseases in principle can be treated by effecting a specific nucleotide change at a specific location in the genome (for example, a C to T change in a specific codon of a gene associated with a disease),⁹ the development of a programmable way to achieve such precision gene editing would represent both a powerful new research tool, as well as a potential new approach to gene editing-based human therapeutics.

SUMMARY OF THE INVENTION

The clustered regularly interspaced short palindromic repeat (CRISPR) system is a recently discovered prokaryotic adaptive immune system¹⁰ that has been modified to enable robust and general genome engineering in a variety of organisms and cell lines.¹¹ CRISPR-Cas (CRISPR associated) systems are protein-RNA complexes that use an RNA molecule (sgRNA) as a guide to localize the complex to a target DNA sequence via base-pairing.¹² In the natural systems, a Cas protein then acts as an endonuclease to cleave the targeted DNA sequence.¹³ The target DNA sequence must be both complementary to the sgRNA, and also contain a “protospacer-adjacent motif” (PAM) at the 3′-end of the complementary region in order for the system to function.¹⁴

Among the known Cas proteins, S. pyogenes Cas9 has been mostly widely used as a tool for genome engineering.¹⁵ This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish nuclease activity, resulting in a dead Cas9 (dCas9) that still retains its ability to bind DNA in a sgRNA-programmed manner.¹⁶ In principle, when fused to another protein or domain, dCas9 can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA.

The potential of the dCas9 complex for genome engineering purposes is immense. Its unique ability to bring proteins to specific sites in a genome programmed by the sgRNA in theory can be developed into a variety of site-specific genome engineering tools beyond nucleases, including transcriptional activators, transcriptional repressors, histone-modifying proteins, integrases, and recombinases.¹¹ Some of these potential applications have recently been implemented through dCas9 fusions with transcriptional activators to afford RNA-guided transcriptional activators,^(17,18) transcriptional repressors,^(16,19,20) and chromatin modification enzymes.²¹ Simple co-expression of these fusions with a variety of sgRNAs results in specific expression of the target genes. These seminal studies have paved the way for the design and construction of readily programmable sequence-specific effectors for the precise manipulation of genomes.

Significantly, 80-90% of protein mutations responsible for human disease arise from the substitution, deletion, or insertion of only a single nucleotide.⁶ Most current strategies for single-base gene correction include engineered nucleases (which rely on the creation of double-strand breaks, DSBs, followed by stochastic, inefficient homology-directed repair, HDR), and DNA-RNA chimeric oligonucleotides.²² The latter strategy involves the design of a RNA/DNA sequence to base pair with a specific sequence in genomic DNA except at the nucleotide to be edited. The resulting mismatch is recognized by the cell's endogenous repair system and fixed, leading to a change in the sequence of either the chimera or the genome. Both of these strategies suffer from low gene editing efficiencies and unwanted gene alterations, as they are subject to both the stochasticity of HDR and the competition between HDR and non-homologous end-joining, NHEJ.²³⁻²⁵ HDR efficiencies vary according to the location of the target gene within the genome,²⁶ the state of the cell cycle,²⁷ and the type of cell/tissue.²⁸ The development of a direct, programmable way to install a specific type of base modification at a precise location in genomic DNA with enzyme-like efficiency and no stochasticity therefore represents a powerful new approach to gene editing-based research tools and human therapeutics.

Some aspects of the disclosure are based on the recognition that certain configurations of a dCas9 domain, and a cytidine deaminase domain fused by a linker are useful for efficiently deaminating target cytidine residues. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with a cytidine deaminase domain fused to the N-terminus of a nuclease inactive Cas9 (dCas9) via a linker was capable of efficiently deaminating target nucleic acids in a double stranded DNA target molecule. See for example, Examples 3 and 4 below, which demonstrate that the fusion proteins, which are also referred to herein as base editors, generate less indels and more efficiently deaminate target nucleic acids than other base editors, such as base editors without a UGI domain. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, where the deaminase domain is fused to the N-terminus of the dCas9 domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7). In some embodiments, the nuclease-inactive Cas9 (dCas9) domain of comprises the amino acid sequence set forth in SEQ ID NO: 263. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 5738). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 275). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 5739-5741).

Some aspects of the disclosure are based on the recognition that certain configurations of a dCas9 domain, and a cytidine deaminase domain fused by a linker are useful for efficiently deaminating target cytidine residues. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain fused to the N-terminus of a nuclease inactive Cas9 (dCas9) via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7) was capable of efficiently deaminating target nucleic acids in a double stranded DNA target molecule. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, where the deaminase domain is fused to the N-terminus of the dCas9 domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7).

In some embodiments, the fusion protein comprises the amino acid residues 11-1629 of the amino acid sequence set forth in SEQ ID NO: 591. In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 591. In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 5737, 5743, 5745, and 5746.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within a subject's genome, e.g., a human's genome. In some embodiments, fusion proteins of Cas9 (e.g., dCas9, nuclease active Cas9, or Cas9 nickase) and deaminases or deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and deaminases or deaminase domains, are provided.

Some aspects of this disclosure provide fusion proteins comprising a Cas9 protein as provided herein that is fused to a second protein (e.g., an enzymatic domain such as a cytidine deaminase domain), thus forming a fusion protein. In some embodiments, the second protein comprises an enzymatic domain, or a binding domain. In some embodiments, the enzymatic domain is a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments, the enzymatic domain is a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). It should be appreciated that the deaminase may be from any suitable organism (e.g., a human or a rat). In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1.

Some aspects of this disclosure provide fusion proteins comprising: (i) a nuclease-inactive Cas9 (dCas9) domain comprising the amino acid sequence of SEQ ID NO: 263; and (ii) an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the dCas9 domain via a linker comprising the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 7). In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 591. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 5737. In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 5738). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 275). In some embodiments, the deaminase is a human APOBEC3G variant of any one of SEQ ID NOs: 5739-5741.

Some aspects of this disclosure provide fusion proteins comprising: (i) a Cas9 nickase domain and (ii) an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the Cas9 nickase domain. In some embodiments, the Cas9 nickase domain comprises a D10X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a histidine at amino acid position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding amino acid position in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises the amino acid sequence as set forth in SEQ ID NO: 267. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1.

Some aspects of this disclosure provide fusion proteins comprising: (i) a Cas9 nickase domain and (ii) an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the Cas9 nickase domain. In some embodiments, the Cas9 nickase domain comprises a D10X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a histidine at amino acid position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding amino acid position in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises the amino acid sequence as set forth in SEQ ID NO: 267. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1.

Other aspects of this disclosure relate to the recognition that fusion proteins comprising a deaminase domain, a dCas9 domain and a uracil glycosylase inhibitor (UGI) domain demonstrate improved efficiency for deaminating target nucleotides in a nucleic acid molecule. Without wishing to be bound by any particular theory, cellular DNA-repair response to the presence of U:G heteroduplex DNA may be responsible for a decrease in nucleobase editing efficiency in cells. Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells, which may initiate base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. As demonstrated herein, Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity. Without wishing to be bound by any particular theory, base excision repair may be inhibited by molecules that bind the single strand, block the edited base, inhibit UGI, inhibit base excision repair, protect the edited base, and/or promote “fixing” of the non-edited strand, etc. Thus, this disclosure contemplates fusion proteins comprising a dCas9-cytidine deaminase domain that is fused to a UGI domain.

In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) domain; a nucleic acid editing domain; and a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the dCas9 domain comprises a D10X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the amino acid sequence of the dCas9 domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the dCas9 domain comprises an H840X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for H. In some embodiments, the amino acid sequence of the dCas9 domain comprises an H840A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the dCas9 domain comprises the amino acid sequence as set forth in SEQ ID NO: 263.

Further aspects of this disclosure relate to the recognition that fusion proteins using a Cas9 nickase as the Cas9 domain demonstrate improved efficiency for editing nucleic acids. For example, aspects of this disclosure relate to the recognition that fusion proteins comprising a Cas9 nickase, a deaminase domain and a UGI domain demonstrate improved efficiency for editing nucleic acids. For example, the improved efficiency for editing nucleotides is described below in the Examples section.

Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. An “indel”, as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels.

Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation, such as a point mutation, in a nucleic acid (e.g. a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations.

In some embodiments, a fusion protein comprises a Cas9 nickase domain, a nucleic acid editing domain; and a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a D10X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a histidine at amino acid position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding amino acid position in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises the amino acid sequence as set forth in SEQ ID NO: 267.

In some embodiments, the deaminase domain of the fusion protein is fused to the N-terminus of the dCas9 domain or the Cas9 nickase. In some embodiments, the UGI domain is fused to the C-terminus of the dCas9 domain or the Cas9 nickase. In some embodiments, the dCas9 domain or the Cas9 nickase and the nucleic acid editing domain are fused via a linker. In some embodiments, the dCas9 domain or the Cas9 nickase and the UGI domain are fused via a linker.

In certain embodiments, linkers may be used to link any of the peptides or peptide domains of the invention. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polpeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may included funtionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

In some embodiments, the linker comprises the amino acid sequence (GGGGS)_(n) (SEQ ID NO: 5), (G)_(n), (EAAAK)_(n) (SEQ ID NO: 6), (GGS)_(n), (SGGS)_(n)(SEQ ID NO: 4288), SGSETPGTSESATPES (SEQ ID NO: 7), (XP)_(n), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)_(n), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7).

In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain]-[optional linker sequence]-[dCas9 or Cas9 nickase]-[optional linker sequence]-[UGI]. In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain]-[optional linker sequence]-[UGI]-[optional linker sequence]-[dCas9 or Cas9 nickase]; [UGI]-[optional linker sequence]-[nucleic acid editing domain]-[optional linker sequence]-[dCas9 or Cas9 nickase]; [UGI]-[optional linker sequence]-[dCas9 or Cas9 nickase]-[optional linker sequence]-[nucleic acid editing domain]; [dCas9 or Cas9 nickase]-[optional linker sequence]-[UGI]-[optional linker sequence]-[nucleic acid editing domain]; or [dCas9 or Cas9 nickase]-[optional linker sequence]-[nucleic acid editing domain]-[optional linker sequence]-[UGI].

In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, or an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a Lamprey CDA1 (pmCDA1) deaminase.

In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is from a human. In some embodiments the deaminase is from a rat. In some embodiments, the deaminase is a rat APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 284). In some embodiments, the deaminase is a human APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 5738). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 275). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 5739-5741). In some embodiments, the deaminase is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 266-284 or 5725-5741.

In some embodiments, the UGI domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 600. In some embodiments, the UGI domain comprises the amino acid sequence as set forth in SEQ ID NO: 600.

Some aspects of this disclosure provide complexes comprising a Cas9 protein or a Cas9 fusion protein as provided herein, and a guide RNA bound to the Cas9 protein or the Cas9 fusion protein.

Some aspects of this disclosure provide methods of using the Cas9 proteins, fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule (a) with a Cas9 protein or a fusion protein as provided herein and with a guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence; or (b) with a Cas9 protein, a Cas9 fusion protein, or a Cas9 protein or fusion protein complex with a gRNA as provided herein.

Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a Cas9 protein or a Cas9 fusion protein as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the kit further comprises an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone.

Some aspects of this disclosure provide polynucleotides encoding a Cas9 protein of a fusion protein as provided herein. Some aspects of this disclosure provide vectors comprising such polynucleotides. In some embodiments, the vector comprises a heterologous promoter driving expression of polynucleotide.

Some aspects of this disclosure provide cells comprising a Cas9 protein, a fusion protein, a nucleic acid molecule, and/or a vector as provided herein.

The description of exemplary embodiments of the reporter systems above is provided for illustration purposes only and not meant to be limiting. Additional reporter systems, e.g., variations of the exemplary systems described in detail above, are also embraced by this disclosure.

The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the deaminase activity of deaminases on single stranded DNA substrates. Single stranded DNA substrates using randomized PAM sequences (NNN PAM) were used as negative controls. Canonical PAM sequences used (NGG PAM)

FIG. 2 shows activity of Cas9:deaminase fusion proteins on single stranded DNA substrates.

FIG. 3 illustrates double stranded DNA substrate binding by Cas9:deaminase:sgRNA complexes.

FIG. 4 illustrates a double stranded DNA deamination assay.

FIG. 5 demonstrates that Cas9 fusions can target positions 3-11 of double-stranded DNA target sequences (numbered according to the schematic in FIG. 5). Upper Gel: 1 μM rAPOBEC1-GGS-dCas9, 125 nM dsDNA, 1 equivalent sgRNA. Mid Gel: 1 μM rAPOBEC1-(GGS)₃ (SEQ ID NO: 596)-dCas9, 125 nM dsDNA, 1 equivalent sgRNA. Lower Gel: 1.85 μM rAPOBEC1-XTEN-dCas9, 125 nM dsDNA, 1 equivalent sgRNA.

FIG. 6 demonstrates that the correct guide RNA, e.g., the correct sgRNA, is required for deaminase activity.

FIG. 7 illustrates the mechanism of target DNA binding of in vivo target sequences by deaminase-dCas9:sgRNA complexes.

FIG. 8 shows successful deamination of exemplary disease-associated target sequences.

FIG. 9 shows in vitro C→T editing efficiencies using His6-rAPOBEC1-XTEN-dCas9.

FIG. 10 shows C→T editing efficiencies in HEK293T cells is greatly enhanced by fusion with UGI.

FIGS. 11A to 11C show NBE1 mediates specific, guide RNA-programmed C to U conversion in vitro. FIG. 11A: Nucleobase editing strategy. DNA with a target C (red) at a locus specified by a guide RNA (green) is bound by dCas9 (blue), which mediates the local denaturation of the DNA substrate. Cytidine deamination by a tethered APOBEC1 enzyme (orange) converts the target C to U. The resulting G:U heteroduplex can be permanently converted to an A:T base pair following DNA replication or repair. If the U is in the template DNA strand, it will also result in an RNA transcript containing a G to A mutation following transcription. FIG. 11B: Deamination assay showing an activity window of approximately five nucleotides. Following incubation of NBE1-sgRNA complexes with dsDNA substrates at 37° C. for 2 h, the 5′ fluorophore-labeled DNA was isolated and incubated with USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37° C. for 1 h to induce DNA cleavage at the site of any uracils. The resulting DNA was resolved on a denaturing polyacrylamide gel, and any fluorophore-linked strands were visualized. Each lane is labeled according to the position of the target C within the protospacer, or with “-” if no target C is present, counting the base distal from the PAM as position 1. FIG. 11C: Deaminase assay showing the sequence specificity and sgRNA-dependence of NBE1. The DNA substrate with a target C at position 7 was incubated with NBE1 as in FIG. 11B with either the correct sgRNA, a mismatched sgRNA, or no sgRNA. No C to U editing is observed with the mismatched sgRNA or with no sgRNA. The positive control sample contains a DNA sequence with a U synthetically incorporated at position 7.

FIGS. 12A to 12B show effects of sequence context and target C position on nucleobase editing efficiency in vitro. FIG. 12A: Effect of changing the sequence surrounding the target C on editing efficiency in vitro. The deamination yield of 80% of targeted strands (40% of total sequencing reads from both strands) for C₇ in the protospacer sequence 5′-TTATTTCGTGGATTTATTTA-3′(SEQ ID NO: 264) was defined as 1.0, and the relative deamination efficiencies of substrates containing all possible single-base mutations at positions 1-6 and 8-13 are shown. Values and error bars reflect the mean and standard deviation of two or more independent biological replicates performed on different days. FIG. 12B: Positional effect of each NC motif on editing efficiency in vitro. Each NC target motif was varied from positions 1 to 8 within the protospacer as indicated in the sequences shown on the right (the PAM shown in red, the protospacer plus one base 5′ to the protospacer are also shown). The percentage of total sequence reads containing T at each of the numbered target C positions following incubation with NBE1 is shown in the graph. Note that the maximum possible deamination yield in vitro is 50% of total sequencing reads (100% of targeted strands). Values and error bars reflect the mean and standard deviation of two or three independent biological replicates performed on different days. FIG. 12B depicts SEQ ID NOs: 285 through 292 from top to bottom, respectively.

FIGS. 13A to 13C show nucleobase editing in human cells. FIG. 13A: Protospacer (black) and PAM (red) sequences of the six mammalian cell genomic loci targeted by nucleobase editors. Target Cs are indicated with subscripted numbers corresponding to their positions within the protospacer. FIG. 13A depicts SEQ ID NOs: 293 through 298 from top to bottom, respectively. FIG. 13B: HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for NBE1, NBE2, and NBE3 at all six genomic loci, and for wt Cas9 with a donor HDR template at three of the six sites (EMX1, HEK293 site 3, and HEK293 site 4). Values and error bars reflect the mean and standard deviation of three independent biological replicates performed on different days. FIG. 13C: Frequency of indel formation, calculated as described in the Methods, is shown following treatment of HEK293T cells with NBE2 and NBE3 for all six genomic loci, or with wt Cas9 and a single-stranded DNA template for HDR at three of the six sites (EMX1, HEK293 site 3, and HEK293 site 4). Values reflect the mean of at least three independent biological replicates performed on different days.

FIGS. 14A to 14C show NBE2- and NBE3-mediated correction of three disease-relevant mutations in mammalian cells. For each site, the sequence of the protospacer is indicated to the right of the name of the mutation, with the PAM highlighted in green and the base responsible for the mutation indicated in bold with a subscripted number corresponding to its position within the protospacer. The amino acid sequence above each disease-associated allele is shown, together with the corrected amino acid sequence following nucleobase editing in red. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were nucleofected with plasmids encoding NBE2 or NBE3 and an appropriate sgRNA. Two days after nucleofection, genomic DNA was extracted and analyzed by HTS to assess pathogenic mutation correction. FIG. 14A: The Alzheimer's disease-associated APOE4 allele is converted to APOE3′ in mouse astrocytes by NBE3 in 11% of total reads (44% of nucleofected astrocytes). Two nearby Cs are also converted to Ts, but with no change to the predicted sequence of the resulting protein (SEQ ID NO: 299). FIG. 14B The cancer-associated p53 N239D mutation is corrected by NBE2 in 11% of treated human lymphoma cells (12% of nucleofected cells) that are heterozygous for the mutation (SEQ ID NO: 300). FIG. 14C The p53 Y163C mutation is corrected by NBE3 in 7.6% of nucleofected human breast cancer cells (SEQ ID NO: 301).

FIGS. 15A to 15D show effects of deaminase-dCas9 linker length and composition on nucleobase editing. Gel-based deaminase assay showing the deamination window of nucleobase editors with deaminase-Cas9 linkers of GGS (FIG. 15A), (GGS)₃ (SEQ ID NO: 596) (FIG. 15B), XTEN (FIG. 15C), or (GGS)₇ (SEQ ID NO: 597) (FIG. 15D). Following incubation of 1.85 μM editor-sgRNA complexes with 125 nM dsDNA substrates at 37° C. for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37° C. for an additional hour to cleave the DNA backbone at the site of any uracils. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with—if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8.

FIGS. 16A to 16B show NBE1 is capable of correcting disease-relevant mutations in vitro. FIG. 16A: Protospacer and PAM sequences (red) of seven disease-relevant mutations. The disease-associated target C in each case is indicated with a subscripted number reflecting its position within the protospacer. For all mutations except both APOE4 SNPs, the target C resides in the template (non-coding) strand. FIG. 16A depicts SEQ ID NOs: 302 through 308 from top to bottom, respectively. FIG. 16B: Deaminase assay showing each dsDNA oligonucleotide before (−) and after (+) incubation with NBE1, DNA isolation, and incubation with USER enzymes to cleave DNA at positions containing U. Positive control lanes from incubation of synthetic oligonucleotides containing U at various positions within the protospacer with USER enzymes are shown with the corresponding number indicating the position of the U.

FIG. 17 shows processivity of NBE1. The protospacer and PAM (red) of a 60-mer DNA oligonucleotide containing eight consecutive Cs is shown at the top. The oligonucleotide (125 nM) was incubated with NBE1 (2 μM) for 2 h at 37° C. The DNA was isolated and analyzed by high-throughput sequencing. Shown are the percent of total reads for the most frequent nine sequences observed. The vast majority of edited strands (>93%) have more than one C converted to T. This figure depicts SEQ ID NO: 309.

FIGS. 18A to 18H show the effect of fusing UGI to NBE1 to generate NBE2. FIG. 18A: Protospacer and PAM (red) sequences of the six mammalian cell genomic loci targeted with nucleobase editors. Editable Cs are indicated with labels corresponding to their positions within the protospacer. FIG. 18A depicts SEQ ID NOs: 293 through 298 from top to bottom, respectively. FIGS. 18B to 18G: HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE1 and UGI, and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for NBE1, NBE1 and UGI, and NBE2 at all six genomic loci. FIG. 18H: C to T mutation rates at 510 Cs surrounding the protospacers of interest for NBE1, NBE1 plus UGI on a separate plasmid, NBE2, and untreated cells are shown. The data show the results of 3,000,000 DNA sequencing reads from 1.5×106 cells. Values reflect the mean of at least two biological experiments conducted on different days.

FIG. 19 shows nucleobase editing efficiencies of NBE2 in U2OS and HEK293T cells. Cellular C to T conversion percentages by NBE2 are shown for each of the six targeted genomic loci in HEK293T cells and U2OS cells. HEK293T cells were transfected using lipofectamine 2000, and U2OS cells were nucleofected. U2OS nucleofection efficiency was 74%. Three days after plasmid delivery, genomic DNA was extracted and analyzed for nucleobase editing at the six genomic loci by FITS. Values and error bars reflect the mean and standard deviation of at least two biological experiments done on different days.

FIG. 20 shows nucleobase editing persists over multiple cell divisions. Cellular C to T conversion percentages by NBE2 are displayed at two genomic loci in HEK293T cells before and after passaging the cells. HEK293T cells were transfected using Lipofectamine 2000. Three days post transfection, the cells were harvested and split in half. One half was subjected to HTS analysis, and the other half was allowed to propagate for approximately five cell divisions, then harvested and subjected to HTS analysis.

FIG. 21 shows genetic variants from ClinVar that can be corrected in principle by nucleobase editing. The NCBI ClinVar database of human genetic variations and their corresponding phenotypes⁶⁸ was searched for genetic diseases that can be corrected by current nucleobase editing technologies. The results were filtered by imposing the successive restrictions listed on the left. The x-axis shows the number of occurrences satisfying that restriction and all above restrictions on a logarithmic scale.

FIG. 22 shows in vitro identification of editable Cs in six genomic loci. Synthetic 80-mers with sequences matching six different genomic sites were incubated with NBE1 then analyzed for nucleobase editing via HTS. For each site, the sequence of the protospacer is indicated to the right of the name of the site, with the PAM highlighted in red. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base. A target C was considered as “editable” if the in vitro conversion efficiency is >10%. Note that maximum yields are 50% of total DNA sequencing reads since the non-targeted strand is not a substrate for nucleobase editing. This figure depicts SEQ ID NOs: 293 through 298 from top to bottom, respectively.

FIG. 23 shows activities of NBE1, NBE2, and NBE3 at EMX1 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the EMX1 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci for the EMX1 sgRNA, as previously determined using the GUIDE-seq method⁵⁵. EMX1 off-target 5 locus did not amplify and is not shown. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 293, and 310 through 318 from top to bottom, respectively.

FIG. 24 shows activities of NBE1, NBE2, and NBE3 at FANCF off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the FANCF sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci for the FANCF sgRNA, as previously determined using the GUIDE-seq method⁵⁵. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 294 and 319 through 326 from top to bottom, respectively.

FIG. 25 shows activities of NBE1, NBE2, and NBE3 at HEK293 site 2 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the HEK293 site 2 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci for the HEK293 site 2 sgRNA, as previously determined using the GUIDE-seq method⁵⁵. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 295, 327, and 328 from top to bottom, respectively.

FIG. 26 shows activities of NBE1, NBE2, and NBE3 at HEK293 site 3 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the HEK293 site 3 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci for the HEK293 site 3 sgRNA, as previously determined using the GUIDE-seq method⁵⁵. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 296 and 659 through 663 from top to bottom, respectively.

FIG. 27 shows activities of NBE1, NBE2, and NBE3 at HEK293 site 4 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the HEK293 site 4 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci for the HEK293 site 4 sgRNA, as previously determined using the GUIDE-seq method⁵⁵. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 297 and 664 through 673 from top to bottom, respectively.

FIG. 28 shows non-target C mutation rates. Shown here are the C to T mutation rates at 2,500 distinct cytosines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8×106 cells.

FIGS. 29A to 29C show base editing in human cells. FIG. 29A shows possible base editing outcomes in mammalian cells. Initial editing resulted in a U:G mismatch. Recognition and excision of the U by uracil DNA glycosylase (UDG) initiated base excision repair (BER), which lead to reversion to the C:G starting state. BER was impeded by BE2 and BE3, which inhibited UDG. The U:G mismatch was also processed by mismatch repair (MMR), which preferentially repaired the nicked strand of a mismatch. BE3 nicked the non-edited strand containing the G, favoring resolution of the U:G mismatch to the desired U:A or T:A outcome. FIG. 29B shows HEK293T cells treated as described in the Materials and Methods in the Examples below. The percentage of total DNA sequencing read with Ts at the target positions indicated show treatment with BE1, BE2, or BE3, or for treatment with wt Cas9 with a donor HDR template. FIG. 29C shows frequency of indel formation following the treatment in FIG. 29B. Values are listed in FIG. 34. For FIGS. 29B and 29C, values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days.

FIGS. 30A to 30B show BE3-mediated correction of two disease-relevant mutations in mammalian cells. The sequence of the protospacer is shown to the right of the mutation, with the PAM in blue and the target base in red with a subscripted number indicating its position within the protospacer. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were treated as described in the Materials and Methods. FIG. 30A shows the Alzheimer's disease-associated APOE4 allele converted to APOE3r in mouse astrocytes by BE3 in 74.9% of total reads. Two nearby Cs were also converted to Ts, but with no change to the predicted sequence of the resulting protein. Identical treatment of these cells with wt Cas9 and donor ssDNA results in only 0.3% correction, with 26.1% indel formation. FIG. 30B shows the cancer associated p53 Y163C mutation corrected by BE3 in 7.6% of nucleofected human breast cancer cells with 0.7% indel formation. Identical treatment of these cells with wt Cas9 and donor ssDNA results in no mutation correction with 6.1% indel formation. This figure depicts SEQ ID NOs: 675 to 680 from top to bottom, respectively.

FIG. 31 shows activities of BE1, BE2, and BE3 at HEK293 site 2 off-targets. HEK293T cells were transfected with plasmids expressing BE1, BE2, or BE3 and a sgRNA matching the HEK293 site 2 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 and dCas9 off-target loci for the HEK293 site 2 sgRNA, as previously determined by Joung and coworkers using the GUIDE-seq method (63), and Adli and coworkers using chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) experiments (18). Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE1, BE2, and BE3. On the far right are displayed the total number of sequencing reads reported, and the ChIP-seq signal intensity reported for each sequence. This figure depicts SEQ ID NOs: 681 to 688 from top to bottom, respectively.

FIG. 32 shows activities of BE1, BE2, and BE3 at HEK293 site 3 off-targets. HEK293T cells were transfected with plasmids expressing BE1, BE2, or BE3 and a sgRNA matching the HEK293 site 3 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci and the top five known dCas9 off-target loci for the HEK293 site 3 sgRNA, as previously determined by Joung and coworkers using the GUIDE-seq method⁵⁴, and using chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) experiments⁶¹. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE1, BE2, and BE3. On the far right are displayed the total number of sequencing reads reported, and the ChIP-seq signal intensity reported for each sequence. This figure depicts SEQ ID NOs: 689 to 699 from top to bottom, respectively.

FIG. 33 shows activities of BE1, BE2, and BE3 at HEK293 site 4 off-targets. HEK293T cells were transfected with plasmids expressing BE1, BE2, or BE3 and a sgRNA matching the HEK293 site 4 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci and the top five known dCas9 off-target loci for the HEK293 site 4 sgRNA, as previously determined using the GUIDE-seq method⁵⁴, and using chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) experiments⁶¹. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE1, BE2, and BE3. On the far right are displayed the total number of sequencing reads reported, and the ChIP-seq signal intensity reported for each sequence. This figure depicts SEQ ID NOs: 700 to 712 from top to bottom, respectively.

FIG. 34 shows mutation rates of non-protospacer bases following BE3-mediated correction of the Alzheimer's disease-associated APOE4 allele to APOE3r in mouse astrocytes. The DNA sequence of the 50 bases on either side of the protospacer from FIG. 30A and FIG. 34B is shown with each base's position relative to the protospacer. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers, with the PAM shown in blue. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base for untreated cells, for cells treated with BE3 and an sgRNA targeting the APOE4 C158R mutation, or for cells treated with BE3 and an sgRNA targeting the VEGFA locus. Neither BE3-treated sample resulted in mutation rates above those of untreated controls. This figure depicts SEQ ID NOs: 713 to 716 from top to bottom, respectively.

FIG. 35 shows mutation rates of non-protospacer bases following BE3-mediated correction of the cancer-associated p53 Y163C mutation in HCC1954 human cells. The DNA sequence of the 50 bases on either side of the protospacer from FIG. 30B and FIG. 39B is shown with each base's position relative to the protospacer. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers, with the PAM shown in blue. Underneath each sequence are the percentages of total sequencing reads with the corresponding base for untreated cells, for cells treated with BE3 and an sgRNA targeting the TP53 Y163C mutation, or for cells treated with BE3 and an sgRNA targeting the VEGFA locus. Neither BE3-treated sample resulted in mutational rates above those of untreated controls. This figure depicts SEQ ID NOs: 717 to 720 from top to bottom, respectively.

FIGS. 36A to 36F show the effects of deaminase, linker length, and linker composition on base editing. FIG. 36A shows a gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, hAPOBEC3G, rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS)₃ (SEQ ID NO: 596)-dCas9, and dCas9-(GGS)₃ (SEQ ID NO: 596)-rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37° C. for 2 hours. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. FIG. 36B shows coomassie-stained denaturing PAGE gel of the expressed and purified proteins used in FIGS. 36C to 36F. FIGS. 36C to 36F show gel-based deaminase assay showing the deamination window of base editors with deaminase-Cas9 linkers of GGS (FIG. 36C), (GGS)₃ (SEQ ID NO: 596) (FIG. 36D), XTEN (FIG. 36E), or (GGS)₇ (SEQ ID NO: 597) (FIG. 36F). Following incubation of 1.85 μM deaminase-dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37° C. for 2 hours, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37° C. for 1 hour to cleave the DNA backbone at the site of any uracils. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with—if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8.

FIGS. 37A to 37C show BE1 base editing efficiencies are dramatically decreased in mammalian cells. FIG. 37A Protospacer (black and red) and PAM (blue) sequences of the six mammalian cell genomic loci targeted by base editors. Target Cs are indicated in red with subscripted numbers corresponding to their positions within the protospacer. FIG. 37B shows synthetic 80-mers with sequences matching six different genomic sites were incubated with BE1 then analyzed for base editing by HTS. For each site, the sequence of the protospacer is indicated to the right of the name of the site, with the PAM highlighted in blue. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base. We considered a target C as “editable” if the in vitro conversion efficiency is >10%. Note that maximum yields are 50% of total DNA sequencing reads since the non-targeted strand is unaffected by BE1. Values are shown from a single experiment. FIG. 37C shows HEK293T cells were transfected with plasmids expressing BE1 and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for BE1 at all six genomic loci. Values and error bars of all data from HEK293T cells reflect the mean and standard deviation of three independent biological replicates performed on different days. FIG. 37A depicts SEQ ID NOs: 721 to 726 from top to bottom, respectively. FIG. 37B depicts SEQ ID NOs: 727 to 732 from top to bottom, respectively.

FIG. 38 shows base editing persists over multiple cell divisions. Cellular C to T conversion percentages by BE2 and BE3 are shown for HEK293 sites 3 and 4 in HEK293T cells before and after passaging the cells. HEK293T cells were nucleofected with plasmids expressing BE2 or BE3 and an sgRNA targeting HEK293 site 3 or 4. Three days after nucleofection, the cells were harvested and split in half. One half was subjected to HTS analysis, and the other half was allowed to propagate for approximately five cell divisions, then harvested and subjected to HTS analysis. Values and error bars reflect the mean and standard deviation of at least two biological experiments.

FIGS. 39A to 39C show non-target C/G mutation rates. Shown here are the C to T and G to A mutation rates at 2,500 distinct cytosines and guanines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8×10⁶ cells. FIGS. 39A and 39B show cellular non-target C to T and G to A conversion percentages by BE1, BE2, and BE3 are plotted individually against their positions relative to a protospacer for all 2,500 cytosines/guanines. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers. FIG. 39C shows average non-target cellular C to T and G to A conversion percentages by BE1, BE2, and BE3 are shown, as well as the highest and lowest individual conversion percentages.

FIGS. 40A to 40B show additional data sets of BE3-mediated correction of two disease-relevant mutations in mammalian cells. For each site, the sequence of the protospacer is indicated to the right of the name of the mutation, with the PAM highlighted in blue and the base responsible for the mutation indicated in red bold with a subscripted number corresponding to its position within the protospacer. The amino acid sequence above each disease-associated allele is shown, together with the corrected amino acid sequence following base editing in green. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were nucleofected with plasmids encoding BE3 and an appropriate sgRNA. Two days after nucleofection, genomic DNA was extracted from the nucleofected cells and analyzed by HTS to assess pathogenic mutation correction. FIG. 40A shows the Alzheimer's disease-associated APOE4 allele is converted to APOE3r in mouse astrocytes by BE3 in 58.3% of total reads only when treated with the correct sgRNA. Two nearby Cs are also converted to Ts, but with no change to the predicted sequence of the resulting protein. Identical treatment of these cells with wt Cas9 and donor ssDNA results in 0.2% correction, with 26.7% indel formation. FIG. 40B shows the cancer-associated p53 Y163C mutation is corrected by BE3 in 3.3% of nucleofected human breast cancer cells only when treated with the correct sgRNA. Identical treatment of these cells with wt Cas9 and donor ssDNA results in no detectable mutation correction with 8.0% indel formation. FIGS. 40A to 40B depict SEQ ID NOs: 733 to 740 from top to bottom, respectively.

FIG. 41 shows a schematic representation of an exemplary USER (Uracil-Specific Excision Reagent) Enzyme-based assay, which may be used to test the activity of various deaminases on single-stranded DNA (ssDNA) substrates.

FIG. 42 is a schematic of the pmCDA-nCas9-UGI-NLS construct and its activity at the HeK-3 site relative to the base editor (rAPOBEC1) and the negative control (untreated).

FIG. 43 is a schematic of the pmCDA1-XTEN-nCas9-UGI-NLS construct and its activity at the HeK-3 site relative to the base editor (rAPOBEC1) and the negative control (untreated).

FIG. 44 shows the percent of total sequencing reads with target C converted to T using cytidine deaminases (CDA) or APOBEC.

FIG. 45 shows the percent of total sequencing reads with target C converted to A using deaminases (CDA) or APOBEC.

FIG. 46 shows the percent of total sequencing reads with target C converted to G using deaminases (CDA) or APOBEC.

FIG. 47 is a schematic of the huAPOBEC3G-XTEN-nCas9-UGI-NLS construct and its activity at the HeK-2 site relative to a mutated form (huAPOBEC3G*(D316R_D317R)-XTEN-nCas9-UGI-NLS, the base editor (rAPOBEC1) and the negative control (untreated).

FIG. 48 shows the schematic of the LacZ construct used in the selection assay of Example 7.

FIG. 49 shows reversion data from different plasmids and constructs.

FIG. 50 shows the verification of lacZ reversion and the purification of reverted clones.

FIG. 51 is a schematic depicting a deamination selection plasmid used in Example 7.

FIG. 52 shows the results of a chloramphenicol reversion assay (pmCDA1 fusion).

FIGS. 53A to 53B demonstrated DNA correction induction of two constructs.

FIG. 54 shows the results of a chloramphenicol reversion assay (huAPOBEC3G fusion).

FIG. 55 shows the activities of BE3 and HF-BE3 at EMX1 off-targets. The sequences, from top to bottom, correspond to SEQ ID NOs: 286-292, 299-301.

FIG. 56 shows on-target base editing efficiencies of BE3 and HF-BE3.

FIG. 57 is a graph demonstrating that mutations affect cytidine deamination with varying degrees. Combinations of mutations that each slightly impairs catalysis allow selective deamination at one position over others. The FANCF site was GGAATC₆C₇C₈TTC₁₁TGCAGCACCTGG (SEQ ID NO: 303).

FIG. 58 is a schematic depicting next generation base editors.

FIG. 59 is a schematic illustrating new base editors made from Cas9 variants.

FIG. 60 shows the base-edited percentage of different NGA PAM sites.

FIG. 61 shows the base-edited percentage of cytidines using NGCG PAM EMX (VRER BE3) and the C₁TC₃C₄C₅ATC₈AC₁₀ATCAACCGGT (SEQ ID NO: 304) spacer.

FIG. 62 shows the based-edited percentages resulting from different NNGRRT PAM sites.

FIG. 63 shows the based-edited percentages resulting from different NNHRRT PAM sites.

FIGS. 64A to 64C show the base-edited percentages resulting from different TTTN PAM sites using Cpf1 BE2. The spacers used were: TTTCCTC₃C₄C₅C₆C₇C₈C₉AC₁₁AGGTAGAACAT (FIG. 64A, SEQ ID NO: 305), TTTCC₁C₂TC₄TGTC₈C₉AC₁₁ACCCTCATCCTG (FIG. 64B, SEQ ID NO: 306), and TTTCC₁C₂C₃AGTC₇C₈TC₁₀C₁₁AC₁₃AC₁₅C₁₆C₁₇TGAAAC (FIG. 64C, SEQ ID NO: 307).

FIG. 65 is a schematic depicting selective deamination as achieved through kinetic modulation of cytidine deaminase point mutagenesis.

FIG. 66 is a graph showing the effect of various mutations on the deamination window probed in cell culture with multiple cytidines in the spacer. The spacer used was: TGC₃C₄C₅C₆TC₈C₉C₁₀TC₁₂C₁₃C₁₄TGGCCC (SEQ ID NO: 308).

FIG. 67 is a graph showing the effect of various mutations on the deamination window probed in cell culture with multiple cytidines in the spacer. The spacer used was: AGAGC₅C₆C₇C₈C₉C₁₀C₁₁TC₁₃AAAGAGA (SEQ ID NO: 309).

FIG. 68 is a graph showing the effect of various mutations on the FANCF site with a limited number of cytidines. The spacer used was: GGAATC₆C₇C₈TTC₁₁TGCAGCACCTGG (SEQ ID NO: 303). Note that the triple mutant (W90Y, R126E, R132E) preferentially edits the cytidine at the sixth position.

FIG. 69 is a graph showing the effect of various mutations on the HEK3 site with a limited number of cytidines. The spacer used was: GGCC₄C₅AGACTGAGCACGTGATGG (SEQ ID NO: 310). Note that the double and triple mutants preferentially edit the cytidine at the fifth position over the cytidine in the fourth position.

FIG. 70 is a graph showing the effect of various mutations on the EMX1 site with a limited number of cytidines. The spacer used was: GAGTC₅C₆GAGCAGAAGAAGAAGGG (SEQ ID NO: 311). Note that the triple mutant only edits the cytidine at the fifth position, not the sixth.

FIG. 71 is a graph showing the effect of various mutations on the HEK2 site with a limited number of cytidines. The spacer used was: GAAC₄AC₆AAAGCATAGACTGCGGG (SEQ ID NO: 312).

FIG. 72 shows on-target base editing efficiencies of BE3 and BE3 comprising mutations W90Y R132E in immortalized astrocytes.

FIG. 73 depicts a schematic of three Cpf1 fusion constructs.

FIG. 74 shows a comparison of plasmid delivery of BE3 and HF-BE3 (EMX1, FANCF, and RNF2).

FIG. 75 shows a comparison of plasmid delivery of BE3 and HF-BE3 (HEK3 and HEK 4).

FIG. 76 shows off-target editing of EMX-1 at all 10 sites.

FIG. 77 shows deaminase protein lipofection to HEK cells using a GAGTCCGAGCAGAAGAAGAAG (SEQ ID NO: 313) spacer. The EMX-1 on-target and EMX-1 off target site 2 were examined.

FIG. 78 shows deaminase protein lipofection to HEK cells using a GGAATCCCTTCTGCAGCACCTGG (SEQ ID NO: 314) spacer. The FANCF on target and FANCF off target site 1 were examined.

FIG. 79 shows deaminase protein lipofection to HEK cells using a GGCCCAGACTGAGCACGTGA (SEQ ID NO: 315) spacer. The HEK-3 on target site was examined.

FIG. 80 shows deaminase protein lipofection to HEK cells using a GGCACTGCGGCTGGAGGTGGGGG (SEQ ID NO: 316) spacer. The HEK-4 on target, off target site 1, site 3, and site 4.

FIG. 81 shows the results of an in vitro assay for sgRNA activity for sgHR_13 (GTCAGGTCGAGGGTTCTGTC (SEQ ID NO: 317) spacer; C8 target: G51 to STOP), sgHR_14 (GGGCCGCAGTATCCTCACTC (SEQ ID NO: 318) spacer; C7 target; C7 target: Q68 to STOP), and sgHR_15 (CCGCCAGTCCCAGTACGGGA (SEQ ID NO: 319) spacer; C10 and C11 are targets: W239 or W237 to STOP).

FIG. 82 shows the results of an in vitro assay for sgHR_17 (CAACCACTGCTCAAAGATGC (SEQ ID NO: 320) spacer; C4 and C5 are targets: W410 to STOP), and sgHR_16 (CTTCCAGGATGAGAACACAG (SEQ ID NO: 321) spacer; C4 and C5 are targets: W273 to STOP).

FIG. 83 shows the direct injection of BE3 protein complexed with sgHR_13 in zebrafish embryos.

FIG. 84 shows the direct injection of BE3 protein complexed with sgHR_16 in zebrafish embryos.

FIG. 85 shows the direct injection of BE3 protein complexed with sgHR_17 in zebrafish embryos.

FIG. 86 shows exemplary nucleic acid changes that may be made using base editors that are capable of making a cytosine to thymine change.

FIG. 87 shows an illustration of apolipoprotein E (APOE) isoforms, demonstrating how a base editor (e.g., BE3) may be used to edit one APOE isoform (e.g., APOE4) into another APOE isoform (e.g., APOE3r) that is associated with a decreased risk of Alzheimer's disease.

FIG. 88 shows base editing of APOE4 to APOE3r in mouse astrocytes.

FIG. 89 shows base editing of PRNP to cause early truncation of the protein at arginine residue 37.

FIG. 90 shows that knocking out UDG (which UGI inhibits) dramatically improves the cleanliness of efficiency of C to T base editing.

FIG. 91 shows that use of a base editor with the nickase but without UGI leads to a mixture of outcomes, with very high indel rates.

FIGS. 92A to 92G show that SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, and VRER-BE3 mediate efficient base editing at target sites containing non-NGG PAMs in human cells. FIG. 92A shows base editor architectures using S. pyogenes and S. aureus Cas9. FIG. 92B shows recently characterized Cas9 variants with alternate or relaxed PAM requirements. FIGS. 92C and 92D show HEK293T cells treated with the base editor variants shown as described in Example 12. The percentage of total DNA sequencing reads (with no enrichment for transfected cells) with C converted to T at the target positions indicated are shown. The PAM sequence of each target tested is shown below the X-axis. The charts show the results for SaBE3 and SaKKH-BE3 at genomic loci with NNGRRT PAMs (FIG. 92C), SaBE3 and SaKKH-BE3 at genomic loci with NNNRRT PAMs (FIG. 92D), VQR-BE3 and EQR-BE3 at genomic loci with NGAG PAMs (FIG. 92E), and with NGAH PAMs (FIG. 92F), and VRER-BE3 at genomic loci with NGCG PAMs (FIG. 92G). Values and error bars reflect the mean and standard deviation of at least two biological replicates.

FIGS. 93A to 93C demonstrate that base editors with mutations in the cytidine deaminase domain exhibit narrowed editing windows. FIGS. 93A to 93C show HEK293T cells transfected with plasmids expressing mutant base editors and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the indicated loci. The percentage of total DNA sequencing reads (without enrichment for transfected cells) with C changed to T at the target positions indicated are shown for the EMX1 site, HEK293 site 3, FANCF site, HEK293 site 2, site A, and site B loci. FIG. 93A illustrates certain cytidine deaminase mutations which narrow the base editing window. See FIG. 98 for the characterization of additional mutations. FIG. 93B shows the effect of cytidine deaminase mutations which effect the editing window width on genomic loci. Combining beneficial mutations has an additive effect on narrowing the editing window. FIG. 93C shows that YE1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 effect the product distribution of base editing, producing predominantly singly-modified products in contrast with BE3. Values and error bars reflect the mean and standard deviation of at least two biological replicates.

FIGS. 94A and 94B show genetic variants from ClinVar that in principle can be corrected by the base editors developed in this work. The NCBI ClinVar database of human genetic variations and their corresponding phenotypes was searched for genetic diseases that in theory can be corrected by base editing. FIG. 94A demonstrates improvement in base editing targeting scope among all pathogenic T→C mutations in the ClinVar database through the use of base editors with altered PAM specificities. The white fractions denote the proportion of pathogenic T-C mutations accessible on the basis of the PAM requirements of either BE3, or BE3 together with the five modified-PAM base editors developed in this work. FIG. 94B shows improvement in base editing targeting scope among all pathogenic T-C mutations in the ClinVar database through the use of base editors with narrowed activity windows. BE3 was assumed to edit Cs in positions 4-8 with comparable efficiency as shown in FIGS. 93A to 93C. YEE-BE3 was assumed to edit with C5>C6>C7>others preference within its activity window. The white fractions denote the proportion of pathogenic T-C mutations that can be edited BE3 without comparable editing of other Cs (left), or that can be edited BE3 or YEE-BE3 without comparable editing of other Cs (right).

FIGS. 95A and 95B show the effect of truncated guide RNAs on base editing window width. HEK293T cells were transfected with plasmids expressing BE3 and sgRNAs of different 5′ truncation lengths. The treated cells were analyzed as described in the Examples. FIG. 95A shows protospacer and PAM sequence (top, SEQ ID NO: 4270) and cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, at a site within the EMX1 genomic locus. At this site, the base editing window was altered through the use of a 17-nt truncated gRNA.

FIG. 95B shows protospacer and PAM sequences (top, SEQ ID NO: 4270) and cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, at sites within the HEK site 3 and site 4 genomic loci. At these sites, no change in the base editing window was observed, but a linear decrease in editing efficiency for all substrate bases as the sgRNA is truncated was noted.

FIG. 96 shows the effect of APOBEC1-Cas9 linker lengths on base editing window width. HEK293T cells were transfected with plasmids expressing base editors with rAPOBEC1-Cas9 linkers of XTEN, GGS, (GGS)₃ (SEQ ID NO: 596), (GGS)₅ (SEQ ID NO: 4271), or (GGS)₇ (SEQ ID NO: 597) and an sgRNA. The treated cells were analyzed as described in the Examples. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for the various base editors with different linkers.

FIGS. 97A to 97C show the effect of rAPOBEC mutations on base editing window width. FIG. 97C shows HEK293T cells transfected with plasmids expressing an sgRNA targeting either Site A or Site B and the BE3 point mutants indicated. The treated cells were analyzed as described in the Examples. All C's in the protospacer and within three basepairs of the protospacer are displayed and the cellular C to T conversion percentages are shown. The ‘editing window widths’, defined as the calculated number of nucleotides within which editing efficiency exceeds the half-maximal value, are displayed for all tested mutants.

FIG. 98 shows the effect of APOBEC1 mutation son product distributions of base editing in mammalian cells. HEK293T cells were transfected with plasmids expressing BE3 or its mutants and an appropriate sgRNAs. The treated cells were analyzed as described in the Examples. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown (left). Percent of total sequencing reads containing the C to T conversion is shown on the right. The BE3 point mutants do not significantly affect base editing efficiencies at HEK site 4, a site with only one target cytidine.

FIG. 99 shows a comparison of on-target editing plasma delivery in BE3 and HF-BE3.

FIG. 100 shows a comparison of on-target editing in protein and plasma delivery of BE3.

FIG. 101 shows a comparison of on-target editing in protein and plasma delivery of HF-BE3.

FIG. 102 shows that both lipofection and installing HF mutations decrease off-target deamination events. The diamond indicates no off targets were detected and the specificity ratio was set to 100.

FIG. 103 shows in vitro C to T editing on a synthetic substrate with Cs placed at even positions in the protospacer (NNNNTC₂TC₄TC₆TC₈TC₁₀TC₁₂TC₁₄TC₁₆TC₁₈TC₂₀NGG, SEQ ID NO: 4272).

FIG. 104 shows in vitro C to T editing on a synthetic substrate with Cs placed at odd positions in the protospacer (NNNNTC₂TC₄TC₆TC₈TC₁₀TC₁₂TC₁₄TC₁₆TC₁₈TC₂₀NGG, SEQ ID NO: 4272).

FIG. 105 includes two graphs depicting the specificity ratio of base editing with plasmid vs. protein delivery.

FIGS. 106A to 106B shows BE3 activity on non-NGG PAM sites. HEK293T cells were transfected with plasmids expressing BE3 and appropriate sgRNA. The treated cells were analyzed as described in the Examples. FIG. 106A shows BE3 activity on sites can be efficiently targeted by SaBE3 or SaKKH-BE3. BE3 shows low but significant activity on the NAG PAM. FIG. 106B shows BE3 has significantly reduced editing at sites with NGA or NGCG PAMs, in contrast to VQR-BE3 or VRER-BE3.

FIGS. 107A to 107B show the effect of APOBEC1 mutations on VQR-BE3 and SaKKH-BE3. HEK293T cells were transfected with plasmids expressing VQR-BE3, SaKKH-BE3 or its mutants and an appropriate sgRNAs. The treated cells were analyzed as described in the Methods. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown. FIG. 107A shows that the window-modulating mutations can be applied to VQR-BE3 to enable selective base editing at sites targetable by NGA PAM. FIG. 107B shows that, when applied to SaKKH-BE3, the mutations cause overall decrease in base editing efficiency without conferring base selectivity within the target window.

FIG. 108 shows a schematic representation of nucleotide editing. The following abbreviations are used: (MMR)—mismatch repair, (BE3 Nickase)—refers to base editor 3, which comprises a Cas9 nickase domain, (UGI)—uracil glycosylase inhibitor, UDG)—uracil DNA glycosylase, (APOBEC)—refers to an APOBEC cytidine deaminase.

DEFINITIONS

As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents.

The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (mc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase.

A nuclease-inactivated Cas9 protein may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.

In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, SEQ ID NO:1 (nucleotide); SEQ ID NO:2 (amino acid)).

(SEQ ID NO: 1) ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGA TGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTT CTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTT TTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCT CGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATT TTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAA GAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTT GGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTAT CATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTA ATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATT GAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAG TTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGT AGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGA TTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTT GGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAAT TTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGAT GATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTG TTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTA AGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAG CGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGA CAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAAC GGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAA TTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGC TCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGA CAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAA ATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAAT AGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGG AATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAA CGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAA CATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTC AAATATGTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAG AAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTT AAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTT GAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCAT GATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAAT GAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGG GGGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAG GTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCT CGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTA GATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATC CATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCT GGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCT GCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTC AAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAA AATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGA ATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCT GTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAA AATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGT GATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCA ATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGAT AACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAA CTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAA GCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGC CAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGAT AGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTT AAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTC CAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCG TATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTT GAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATG ATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTT TACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGA GAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATT GTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATG CCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCC AAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAA AAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCT TATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTA AAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTT GAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAA AAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAAC GGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAG CTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTAT GAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTG GAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTT TCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCA TATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATT CATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTT GATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGAT GCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGAT TTGAGTCAGCTAGGAGGTGACTGA (SEQ ID NO: 2) MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGAL LFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRL IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINAS RVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (single underline: HNH domain; double underline: RuvC domain)

In some embodiments, wild type Cas9 corresponds to, or comprises SEQ ID NO:3 (nucleotide) and/or SEQ ID NO: 4 (amino acid):

(SEQ ID NO: 3) ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGA TGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTG TTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTC CTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCT CGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATT TTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAA GAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTT GGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTAT CACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTA ATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATT GAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAG TTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGT GGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGG CTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTC GGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAAC TTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGAT GACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTA TTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTG AGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAA AGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGT CAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAAC GGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAG TTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTA AAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGT AGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGG CAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAA ATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAAC TCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGG AATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAG AGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAG CACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTT AAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAG AAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTT AAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTC GAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCAT GACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAAT GAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGG GAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAG GTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCG CGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTC GATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATC CATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCC GGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCA GCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTT AAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGC GAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAG AGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCAT CCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTA CAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTA TCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGAT TCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGT GACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGG CAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACT AAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAA CGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTA GATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAA GTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGAT TTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGAC GCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAG CTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAG ATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTC TTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAAC GGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAA ATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCC ATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTT TCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGT AAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTT GCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAA CTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCT TTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTA AAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAA AATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAAC GAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCAT TACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTT GTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAA TTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGC GCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATT ATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTAT TTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTA GACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATA GATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTC TCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGAT TACAAGGATGACGATGACAAGGCTGCAGGA (SEQ ID NO: 4) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (single underline: HNH domain; double underline: RuvC domain)

In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2, SEQ ID NO: 8 (nucleotide); and Uniport Reference Sequence: Q99ZW2, SEQ ID NO: 10 (amino acid).

(SEQ ID NO: 8) ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGA TGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTT CTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTT TTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCT CGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATT TTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAA GAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTT GGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTAT CATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTA ATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATT GAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAG TTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGT GGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGA TTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTT GGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAAT TTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGAT GATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTG TTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTA AGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAA CGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGA CAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAAC GGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAA TTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGC TCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGA CAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAA ATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAAT AGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGG AATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAA CGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAA CATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTC AAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAG AAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTT AAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTT GAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCAT GATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAAT GAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGG GAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAG GTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCT CGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTA GATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATC CATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCT GGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCT GCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTC AAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGT GAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAA CGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCAT CCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTC CAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTA AGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGAT TCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCG GATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGA CAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACG AAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAA CGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTG GATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAG GTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGAT TTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGAT GCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAA CTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAA ATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTC TTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAAT GGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAA ATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCC ATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTC TCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGT AAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTA GCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAG TTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCC TTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTT AAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAA AACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAAT GAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCAT TATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTT GTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAA TTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGT GCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATT ATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATAT TTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTA GATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATT GATTTGAGTCAGCTAGGAGGTGACTGA (SEQ ID NO: 10) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (single underline: HNH domain; double underline: RuvC domain)

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria. meningitidis (NCBI Ref: YP_002342100.1) or to a Cas9 from any of the organisms listed in Example 5.

In some embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and/or H840A mutation.

dCas9 (D10A and H840A):

(SEQ ID NO: 9) MDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS GET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLI EGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIEC FDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN

NTQLCINEKLYLYYLCINGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKV

TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (single underline: HNH domain; double underline: RuvC domain).

In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided in SEQ ID NO: 10, or at corresponding positions in any of the amino acid sequences provided in SEQ ID NOs: 11-260. Without wishing to be bound by any particular theory, the presence of the catalytic residue H840 restores the activity of the Cas9 to cleave the non-edited (e.g., non-deaminated) strand containing a G opposite the targeted C. Restoration of H840 (e.g., from A840) does not result in the cleavage of the target strand containing the C. Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a G to A change on the non-edited strand. A schematic representation of this process is shown in FIG. 108. Briefly, the C of a C-G basepair can be deaminated to a U by a deaminase, e.g., an APOBEC deamonase. Nicking the non-edited strand, having the G, facilitates removal of the G via mismatch repair mechanisms. UGI inhibits UDG, which prevents removal of the U.

In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 (e.g., variants of SEQ ID NO: 10) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO: 10. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 10) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 10, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.

In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art.

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); or Neisseria. meningitidis (NCBI Ref: YP_002342100.1).

The term “deaminase” or “deaminase domain,” as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase or deaminase domain is a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism, that does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase from an organism.

The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a nuclease may refer to the amount of the nuclease that is sufficient to induce cleavage of a target site specifically bound and cleaved by the nuclease. In some embodiments, an effective amount of a fusion protein provided herein, e.g., of a fusion protein comprising a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain) may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nuclease, a deaminase, a recombinase, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used.

The term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain). In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic-acid editing protein. In some embodiments, a linker joins a dCas9 and a nucleic-acid editing protein. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^(th) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

The term “nucleic acid editing domain,” as used herein refers to a protein or enzyme capable of making one or more modifications (e.g., deamination of a cytidine residue) to a nucleic acid (e.g., DNA or RNA). Exemplary nucleic acid editing domains include, but are not limited to a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments the nucleic acid editing domain is a deaminase (e.g., a cytidine deaminase, such as an APOBEC or an AID deaminase).

The term “proliferative disease,” as used herein, refers to any disease in which cell or tissue homeostasis is disturbed in that a cell or cell population exhibits an abnormally elevated proliferation rate. Proliferative diseases include hyperproliferative diseases, such as pre-neoplastic hyperplastic conditions and neoplastic diseases. Neoplastic diseases are characterized by an abnormal proliferation of cells and include both benign and malignant neoplasias. Malignant neoplasia is also referred to as cancer.

The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^(th) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

The term “RNA-programmable nuclease,” and “RNA-guided nuclease” are used interchangeably herein and refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application, U.S. Ser. No. 61/874,682, filed Sep. 6, 2013, entitled “Switchable Cas9 Nucleases And Uses Thereof,” and U.S. Provisional Patent Application, U.S. Ser. No. 61/874,746, filed Sep. 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.” For example, an extended gRNA will, e.g., bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. In some embodiments, the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example Cas9 (Csn1) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference.

Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins are able to be targeted, in principle, to any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.

The term “target site” refers to a sequence within a nucleic acid molecule that is deaminated by a deaminase or a fusion protein comprising a deaminase, (e.g., a dCas9-deaminase fusion protein provided herein).

The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

The term “nucleobase editors (NBEs)” or “base editors (BEs),” as used herein, refers to the Cas9 fusion proteins described herein. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 fused to a deaminase and further fused to a UGI domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and further fused to a UGI domain. In some embodiments, the dCas9 of the fusion protein comprises a D10A and a H840A mutation of SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260, which inactivates nuclease activity of the Cas9 protein. In some embodiments, the fusion protein comprises a D10A mutation and comprises a histidine at residue 840 of SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260, which renders Cas9 capable of cleaving only one strand of a nucleic acid duplex. An example of a Cas9 nickase is shown below in SEQ ID NO: 674. The terms “nucleobase editors (NBEs)” and “base editors (BEs)” may be used interchangeably.

The term “uracil glycosylase inhibitor” or “UGI,” as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme.

The term “Cas9 nickase,” as used herein, refers to a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments, a Cas9 nickase comprises a D10A mutation and has a histidine at position H840 of SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260. For example, a Cas9 nickase may comprise the amino acid sequence as set forth in SEQ ID NO: 674. Such a Cas9 nickase has an active HNH nuclease domain and is able to cleave the non-targeted strand of DNA, i.e., the strand bound by the gRNA. Further, such a Cas9 nickase has an inactive RuvC nuclease domain and is not able to cleave the targeted strand of the DNA, i.e., the strand where base editing is desired.

Exemplary Cas9 nickase (Cloning vector pPlatTET-gRNA2; Accession No. BAV54124).

(SEQ ID NO: 674) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Some aspects of this disclosure provide fusion proteins that comprise a domain capable of binding to a nucleotide sequence (e.g., a Cas9, or a Cpf1 protein) and an enzyme domain, for example, a DNA-editing domain, such as, e.g., a deaminase domain. The deamination of a nucleobase by a deaminase can lead to a point mutation at the respective residue, which is referred to herein as nucleic acid editing. Fusion proteins comprising a Cas9 variant or domain and a DNA editing domain can thus be used for the targeted editing of nucleic acid sequences. Such fusion proteins are useful for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals; for the introduction of targeted mutations, e.g., for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently re-introduced into the same or another subject; and for the introduction of targeted mutations, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a subject. Typically, the Cas9 domain of the fusion proteins described herein does not have any nuclease activity but instead is a Cas9 fragment or a dCas9 protein or domain. Methods for the use of Cas9 fusion proteins as described herein are also provided.

Cas9 Domains of Nucleobase Editors

Non-limiting, exemplary Cas9 domains are provided herein. The Cas9 domain may be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase. In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain may be a Cas9 domain that cuts both strands of a duplexed nucleic acid (e.g., both strands of a duplexed DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences as set forth in SEQ ID NOs: 10-263. In some embodiments the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 10-263. In some embodiments, the Cas9 domain comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 10-263. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NOs: 10-263.

In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain may bind to a duplexed nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the duplexed nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation of the amino acid sequence set forth in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation of the amino acid sequence set forth in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. As one example, a nuclease-inactive Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 263 (Cloning vector pPlatTET-gRNA2, Accession No. BAV54124).

(SEQ ID NO: 263 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD; see, e.g., Qi et al., Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013; 152(5):1173-83, the entire contents of which are incorporated herein by reference).

Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference). In some embodiments the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequences that has 1,2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 10-263. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at leat 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NOs: 10-263.

In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments the Cas9 nickase cleaves the target strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base paired to (complementary to) a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises a D10A mutation and has a histidine at position 840 of SEQ ID NO: 10, or a mutation in any of SEQ ID NOs: 11-260. For example, a Cas9 nickase may comprise the amino acid sequence as set forth in SEQ ID NO: 674. In some embodiments the Cas9 nickase cleaves the non-target, non-base-edited strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base paired to a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10 of SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260. In some embodiments the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure.

Cas9 Domains with Reduced PAM Exclusivity

Some aspects of the disclosure provide Cas9 domains that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed at a precise location, for example where a target base is placed within a 4 base region (e.g., a “deamination window”), which is approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.

In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises the amino acid sequence SEQ ID NO: 4273. In some embodiments, the SaCas9 comprises a N579X mutation of SEQ ID NO: 4273, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for N. In some embodiments, the SaCas9 comprises a N579A mutation of SEQ ID NO: 4273, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of a E781X, a N967X, and a R1014X mutation of SEQ ID NO: 4273, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of a E781K, a N967K, and a R1014H mutation of SEQ ID NO: 4273, or one or more corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SaCas9 domain comprises a E781K, a N967K, or a R1014H mutation of SEQ ID NO: 4273, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260.

In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 4273-4275. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 4273-4275. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any one of SEQ ID NOs: 4273-4275.

Exemplary SaCas9 sequence (SEQ ID NO: 4273) KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRG ARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEE EFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQ LERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLL ETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY NALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNE EDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIY QSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWH TNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKV INAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRT TGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRS VSFDNSFNNKVLVKQEE N SKKGNRTPFQYLSSSDSKISYETFKKHILNLAK GKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYF RVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIF KEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDF KDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLK KLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKY SKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLD NGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNND LIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIA SKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG Residue N579 of SEQ ID NO: 4273, which is underlined and in bold, may be mutated (e.g., to a A579) to yield a SaCas9 nickase.

Exemplary SaCas9n sequence (SEQ ID NO: 4274) KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF NYEVDHIIPRSVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISY ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK KG. Residue A579 of SEQ ID NO: xx, which can be mutated from N579 of SEQ ID NO: 4274 to yield a SaCas9 nickase, is underlined and in bold.

Exemplary SaKKH Cas9 (SEQ ID NO: 4275) KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF NYEVDHIIPRSVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISY ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK EIFITPHQIKHIKDFKDYKYSHRVDKKPNR K LINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK KLKKISNQAEFIASFY K NDLIKINGELYRVIGVNNDLLNRIEVNMIDITY REYLENMNDKRPP H IIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK KG. Residue A579 of SEQ ID NO: 4275, which can be mutated from N579 of SEQ ID NO: 4275 to yield a SaCas9 nickase, is underlined and in bold. Residues K781, K967, and H1014 of SEQ ID NO: 4275, which can be mutated from E781, N967, and R1014 of SEQ ID NO: 4275 to yield a SaKKH Cas9 are underlined and in italics.

In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcus pyogenes (SpCas9). In some embodiments, the SpCas9 domain is a nuclease active SpCas9, a nuclease inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 comprises the amino acid sequence SEQ ID NO: 4276. In some embodiments, the SpCas9 comprises a D9X mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the SpCas9 comprises a D9A mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a NGG, a NGA, or a NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a R1334X, and a T1336X mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134E, R1334Q, and T1336R mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises a D1134E, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a R1334X, and a T1336X mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134V, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises a D1134V, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a G1217X, a R1334X, and a T1336X mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134V, a G1217R, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises a D1134V, a G1217R, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260.

In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 4276-4280. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 4276-4280. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any one of SEQ ID NOs: 4276-4280.

Exemplary SpCas9 (SEQ ID NO: 4276) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILR VNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Exemplary SpCas9n (SEQ ID NO: 4277) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILR VNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Exemplary SpEQR Cas9 (SEQ ID NO: 4278) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILR VNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK KDWDPKKYGGF E SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF DTTIDRK Q Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD Residues E1134, Q1334, and R1336 of SEQ ID NO: 4278, which can be mutated from D1134, R1334, and T1336 of SEQ ID NO: 4278 to yield a SpEQR Cas9, are underlined and in bold.

Exemplary SpVQR Cas9 (SEQ ID NO: 4279) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQS ITGLYETRIDLSQLGGD Residues V1134, Q1334, and R1336 of SEQ ID NO: 4279, which can be mutated from D1134, R1334, and T1336 of SEQ ID NO: 4279 to yield a SpVQR Cas9, are underlined and in bold.

Exemplary SpVRER Cas9 (SEQ ID NO: 4280) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK E Y R STKEVLDATLIHQS ITGLYETRIDLSQLGGD Residues V1134, R1217, Q1334, and R1336 of SEQ ID NO: 4280, which can be mutated from D1134, G1217, R1334, and T1336 of SEQ ID NO: 4280 to yield a SpVRER Cas9, are underlined and in bold.

The following are exemplary fusion proteins (e.g., base editing proteins) capable of binding to a nucleic acid sequence having a non-canonical (e.g., a non-NGG) PAM sequence:

Exemplary SaBE3 (rAPOBEC1-XTEN-SaCas9n-UGI-NLS) (SEQ ID NO: 4281) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESKRNYILG LDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLL FDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELST KEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSF IDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN DLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEF TNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPV VKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTT GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLV KQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIK DFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKL LMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAH LDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKL KKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI IKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGSGGSTNLSDIIEKETGKQLVIQESIL MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG GSPKKKRKV Exemplary SaKKH-BE3 (rAPOBEC1-XTEN-SaCas9n-UGI-NLS) (SEQ ID NO: 4282) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESKRNYILG LDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLL FDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELST KEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSF IDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN DLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEF TNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPV VKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTT GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLV KQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIK DFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKL LMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAH LDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKL KKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHI IKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGSGGSTNLSDIIEKETGKQLVIQESIL MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG GSPKKKRKV Exemplary EQR-BE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS) (SEQ ID NO: 4283) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIG LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAE DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFESPTVAYS VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI DRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESIL MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG GSPKKKRKV VQR-BE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS) (SEQ ID NO: 4284) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIG LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAE DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYS VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI DRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESIL MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG GSPKKKRKV VRER-BE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS) (SEQ ID NO: 4285) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIG LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAE DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYS VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI DRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESIL MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG GSPKKKRKV

High Fidelity Base Editors

Some aspects of the disclosure provide Cas9 fusion proteins (e.g., any of the fusion proteins provided herein) comprising a Cas9 domain that has high fidelity. Additional aspects of the disclosure provide Cas9 fusion proteins (e.g., any of the fusion proteins provided herein) comprising a Cas9 domain with decreased electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA, as compared to a wild-type Cas9 domain. In some embodiments, a Cas9 domain (e.g., a wild type Cas9 domain) comprises one or more mutations that decreases the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of a N497X, a R661X, a Q695X, and/or a Q926X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of a N497A, a R661A, a Q695A, and/or a Q926A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the Cas9 domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the Cas9 domain (e.g., of any of the fusion proteins provided herein) comprises the amino acid sequence as set forth in SEQ ID NO: 325. In some embodiments, the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 285. Cas9 domains with high fidelity are known in the art and would be apparent to the skilled artisan. For example, Cas9 domains with high fidelity have been described in Kleinstiver, B. P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and Slaymaker, I. M., et al. “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015); the entire contents of each are incorporated herein by reference.

It should be appreciated that the base editors provided herein, for example base editor 2 (BE2) or base editor 3 (BE3), may be converted into high fidelity base editors by modifying the Cas9 domain as described herein to generate high fidelity base editors, for example high fidelity base editor 2 (HF-BE2) or high fidelity base editor 3 (HF-BE3). In some embodiments, base editor 2 (BE2) comprises a deaminase domain, a dCas9, and a UGI domain. In some embodiments, base editor 3 (BE3) comprises a deaminase domain an nCas9 domain and a UGI domain.

Cas9 domain where mutations relative to Cas9 of SEQ ID NO: 10 are shown in bold and underlines (SEQ ID NO: 325) DKKYSIGL A IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT A FDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWG A LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM A LIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETR A ITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGD HF-BE3 (SEQ ID NO: 285) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEK VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY TGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY ETRIDLSQLGGD

Cas9 Fusion Proteins

Any of the Cas9 domains (e.g., a nuclease active Cas9 protein, a nuclease-inactive dCas9 protein, or a Cas9 nickase protein) disclosed herein may be fused to a second protein, thus fusion proteins provided herein comprise a Cas9 domain as provided herein and a second protein, or a “fusion partner”. In some embodiments, the second protein is fused to the N-terminus of the Cas9 domain. However, in other embodiments, the second protein is fused to the C-terminus of the Cas9 domain. In some embodiments, the second protein that is fused to the Cas9 domain is a nucleic acid editing domain. In some embodiments, the Cas9 domain and the nucleic acid editing domain are fused via a linker, while in other embodiments the Cas9 domain and the nucleic acid editing domain are fused directly to one another. In some embodiments, the linker comprises (GGGS)_(n) (SEQ ID NO: 265), (GGGGS)_(n) (SEQ ID NO: 5), (G)_(n), (EAAAK)_(n) (SEQ ID NO: 6), (GGS)_(n), (SGGS)_(n)(SEQ ID NO: 4288), SGSETPGTSESATPES (SEQ ID NO: 7), or (XP)_(n) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises a (GGS)_(n) motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (GGS)_(n) motif, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises an amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 7), also referred to as the XTEN linker in the Examples). The length of the linker can influence the base to be edited, as illustrated in the Examples. For example, a linker of 3-amino-acid long (e.g., (GGS)₁) may give a 2-5, 2-4, 2-3, 3-4 base editing window relative to the PAM sequence, while a 9-amino-acid linker (e.g., (GGS)₃ (SEQ ID NO: 596)) may give a 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6 base editing window relative to the PAM sequence. A 16-amino-acid linker (e.g., the XTEN linker) may give a 2-7, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, 4-7, 4-6, 4-5, 5-7, 5-6, 6-7 base window relative to the PAM sequence with exceptionally strong activity, and a 21-amino-acid linker (e.g., (GGS)₇ (SEQ ID NO: 597)) may give a 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, 7-8 base editing window relative to the PAM sequence. The novel finding that varying linker length may allow the dCas9 fusion proteins of the disclosure to edit nucleobases different distances from the PAM sequence affords significant clinical importance, since a PAM sequence may be of varying distance to the disease-causing mutation to be corrected in a gene. It is to be understood that the linker lengths described as examples here are not meant to be limiting.

In some embodiments, the second protein comprises an enzymatic domain. In some embodiments, the enzymatic domain is a nucleic acid editing domain. Such a nucleic acid editing domain may be, without limitation, a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, or an acetyltransferase. Non-limiting exemplary binding domains that may be used in accordance with this disclosure include transcriptional activator domains and transcriptional repressor domains.

Deaminase Domains

In some embodiments, second protein comprises a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain can catalyze a C to U base change. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytidine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, e.g., rAPOBEC1. In some embodiments, the deaminase is a Petromyzon marinus cytidine deaminase 1 (pmCDA1). In some embodiments, the deaminase is a human APOBEC3G (SEQ ID NO: 275). In some embodiments, the deaminase is a fragment of the human APOBEC3G (SEQ ID NO: 5740). In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R_D317R mutation (SEQ ID NO: 5739). In some embodiments, the deaminase is a frantment of the human APOBEC3G and comprising mutations corresponding to the D316R_D317R mutations in SEQ ID NO: 275 (SEQ ID NO: 5741).

In some embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the deaminase domain of any one of SEQ ID NOs: 266-284, 607-610, 5724-5736, or 5738-5741. In some embodiments, the nucleic acid editing domain comprises the amino acid sequence of any one of SEQ ID NOs: 266-284, 607-610, 5724-5736, or 5738-5741.

Deaminase Domains that Modulate the Editing Window of Base Editors

Some aspects of the disclosure are based on the recognition that modulating the deaminase domain catalytic activity of any of the fusion proteins provided herein, for example by making point mutations in the deaminase domain, affect the processivity of the fusion proteins (e.g., base editors). For example, mutations that reduce, but do not eliminate, the catalytic activity of a deaminase domain within a base editing fusion protein can make it less likely that the deaminase domain will catalyze the deamination of a residue adjacent to a target residue, thereby narrowing the deamination window. The ability to narrow the deamination window may prevent unwanted deamination of residues adjacent of specific target residues, which may decrease or prevent off-target effects.

In some embodiments, any of the fusion proteins provided herein comprise a deaminase domain (e.g., a cytidine deaminase domain) that has reduced catalytic deaminase activity. In some embodiments, any of the fusion proteins provided herein comprise a deaminase domain (e.g., a cytidine deaminase domain) that has a reduced catalytic deaminase activity as compared to an appropriate control. For example, the appropriate control may be the deaminase activity of the deaminase prior to introducing one or more mutations into the deaminase. In other embodiments, the appropriate control may be a wild-type deaminase. In some embodiments, the appropriate control is a wild-type apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the appropriate control is an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, or an APOBEC3H deaminase. In some embodiments, the appropriate control is an activation induced deaminase (AID). In some embodiments, the appropriate control is a cytidine deaminase 1 from Petromyzon marinus (pmCDA1). In some embodiments, the deaminase domain may be a deaminase domain that has at least 1%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% less catalytic deaminase activity as compared to an appropriate control.

In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase.

In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X, R326X of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of D316R, D317R, R320A, R320E, R313A, W285A, W285Y, R326E of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase.

In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a H121R and a H122R mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R126A mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R126E mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R118A mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90A mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R132E mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y and a R126E mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R126E and a R132E mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y and a R132E mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y, R126E, and R132E mutation of rAPOBEC1 (SEQ ID NO: 284), or one or more corresponding mutations in another APOBEC deaminase.

In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a D316R and a D317R mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320A mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320E mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R313A mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285A mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R326E mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y and a R320E mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320E and a R326E mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y and a R326E mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y, R320E, and R326E mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase.

Some aspects of this disclosure provide fusion proteins comprising (i) a nuclease-inactive Cas9 domain; and (ii) a nucleic acid editing domain. In some embodiments, a nuclease-inactive Cas9 domain (dCas9), comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 as provided by any one of SEQ ID NOs: 10-263, and comprises mutations that inactivate the nuclease activity of Cas9. Mutations that render the nuclease domains of Cas9 inactive are well-known in the art. For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)). In some embodiments, the dCas9 of this disclosure comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the dCas9 of this disclosure comprises a H840A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the dCas9 of this disclosure comprises both D10A and H840A mutations of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the Cas9 further comprises a histidine residue at position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. The presence of the catalytic residue H840 restores the activity of the Cas9 to cleave the non-edited strand containing a G opposite the targeted C. Restoration of H840 does not result in the cleavage of the target strand containing the C. In some embodiments, the dCas9 comprises an amino acid sequence of SEQ ID NO: 263. It is to be understood that other mutations that inactivate the nuclease domains of Cas9 may also be included in the dCas9 of this disclosure.

The Cas9 or dCas9 domains comprising the mutations disclosed herein, may be a full-length Cas9, or a fragment thereof. In some embodiments, proteins comprising Cas9, or fragments thereof, are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9, e.g., a Cas9 comprising the amino acid sequence of SEQ ID NO: 10.

Any of the Cas9 fusion proteins of this disclosure may further comprise a nucleic acid editing domain (e.g., an enzyme that is capable of modifying nucleic acid, such as a deaminase). In some embodiments, the nucleic acid editing domain is a DNA-editing domain. In some embodiments, the nucleic acid editing domain has deaminase activity. In some embodiments, the nucleic acid editing domain comprises or consists of a deaminase or deaminase domain. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 family deaminase. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). Some nucleic-acid editing domains as well as Cas9 fusion proteins including such domains are described in detail herein. Additional suitable nucleic acid editing domains will be apparent to the skilled artisan based on this disclosure and knowledge in the field.

Some aspects of the disclosure provide a fusion protein comprising a Cas9 domain fused to a nucleic acid editing domain, wherein the nucleic acid editing domain is fused to the N-terminus of the Cas9 domain. In some embodiments, the Cas9 domain and the nucleic acid editing-editing domain are fused via a linker. In some embodiments, the linker comprises a (GGGS)_(n)(SEQ ID NO: 265), a (GGGGS)_(n) (SEQ ID NO: 5), a (G)_(n), an (EAAAK)_(n) (SEQ ID NO: 6), a (GGS)_(n), (SGGS)_(n)(SEQ ID NO: 4288), an SGSETPGTSESATPES (SEQ ID NO: 7) motif (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference), or an (XP)_(n) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. In some embodiments, n is independently 1,2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or, if more than one linker or more than one linker motif is present, any combination thereof. In some embodiments, the linker comprises a (GGS)_(n) motif, wherein n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the linker comprises a (GGS)_(n) motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7). Additional suitable linker motifs and linker configurations will be apparent to those of skill in the art. In some embodiments, suitable linker motifs and configurations include those described in Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10):1357-69, the entire contents of which are incorporated herein by reference. Additional suitable linker sequences will be apparent to those of skill in the art based on the instant disclosure. In some embodiments, the general architecture of exemplary Cas9 fusion proteins provided herein comprises the structure:

-   -   [NH₂]-[nucleic acid editing domain]-[Cas9]-[COOH] or     -   [NH₂]-[nucleic acid editing domain]-[linker]-[Cas9]-[COOH],         wherein NH₂ is the N-terminus of the fusion protein, and COOH is         the C-terminus of the fusion protein.

The fusion proteins of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein comprises a nuclear localization sequence (NLS). In some embodiments, the NLS of the fusion protein is localized between the nucleic acid editing domain and the Cas9 domain. In some embodiments, the NLS of the fusion protein is localized C-terminal to the Cas9 domain.

Other exemplary features that may be present are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.

In some embodiments, the nucleic acid editing domain is a deaminase. For example, in some embodiments, the general architecture of exemplary Cas9 fusion proteins with a deaminase domain comprises the structure:

-   -   [NH₂]-[NLS]-[deaminase]-[Cas9]-[COOH],     -   [NH2]-[Cas9]-[deaminase]-[COOH],     -   [NH₂]-[deaminase]-[Cas9]-[COOH], or     -   [NH₂]-[deaminase]-[Cas9]-[NLS]-[COOH]         wherein NLS is a nuclear localization sequence, NH₂ is the         N-terminus of the fusion protein, and COOH is the C-terminus of         the fusion protein. Nuclear localization sequences are known in         the art and would be apparent to the skilled artisan. For         example, NLS sequences are described in Plank et al.,         PCT/EP2000/011690, the contents of which are incorporated herein         by reference for their disclosure of exemplary nuclear         localization sequences. In some embodiments, a NLS comprises the         amino acid sequence PKKKRKV (SEQ ID NO: 741) or         MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 742). In some         embodiments, a linker is inserted between the Cas9 and the         deaminase. In some embodiments, the NLS is located C-terminal of         the Cas9 domain. In some embodiments, the NLS is located         N-terminal of the Cas9 domain. In some embodiments, the NLS is         located between the deaminase and the Cas9 domain. In some         embodiments, the NLS is located N-terminal of the deaminase         domain. In some embodiments, the NLS is located C-terminal of         the deaminase domain.

One exemplary suitable type of nucleic acid editing domain is a cytidine deaminase, for example, of the APOBEC family. The apolipoprotein B mRNA-editing complex (APOBEC) family of cytidine deaminase enzymes encompasses eleven proteins that serve to initiate mutagenesis in a controlled and beneficial manner.²⁹ One family member, activation-induced cytidine deaminase (AID), is responsible for the maturation of antibodies by converting cytosines in ssDNA to uracils in a transcription-dependent, strand-biased fashion.³⁰ The apolipoprotein B editing complex 3 (APOBEC3) enzyme provides protection to human cells against a certain HIV-1 strain via the deamination of cytosines in reverse-transcribed viral ssDNA.³¹ These proteins all require a Zn²⁺-coordinating motif (His-X-Glu-X₂₃₋₂₆-Pro-Cys-X₂₋₄-Cys; SEQ ID NO: 598) and bound water molecule for catalytic activity. The Glu residue acts to activate the water molecule to a zinc hydroxide for nucleophilic attack in the deamination reaction. Each family member preferentially deaminates at its own particular “hotspot”, ranging from WRC (W is A or T, R is A or G) for hAID, to TTC for hAPOBEC3F.³² A recent crystal structure of the catalytic domain of APOBEC3G revealed a secondary structure comprised of a five-stranded β-sheet core flanked by six α-helices, which is believed to be conserved across the entire family.³³ The active center loops have been shown to be responsible for both ssDNA binding and in determining “hotspot” identity.³⁴ Overexpression of these enzymes has been linked to genomic instability and cancer, thus highlighting the importance of sequence-specific targeting.³⁵

Some aspects of this disclosure relate to the recognition that the activity of cytidine deaminase enzymes such as APOBEC enzymes can be directed to a specific site in genomic DNA. Without wishing to be bound by any particular theory, advantages of using Cas9 as a recognition agent include (1) the sequence specificity of Cas9 can be easily altered by simply changing the sgRNA sequence; and (2) Cas9 binds to its target sequence by denaturing the dsDNA, resulting in a stretch of DNA that is single-stranded and therefore a viable substrate for the deaminase. It should be understood that other catalytic domains, or catalytic domains from other deaminases, can also be used to generate fusion proteins with Cas9, and that the disclosure is not limited in this regard.

Some aspects of this disclosure are based on the recognition that Cas9:deaminase fusion proteins can efficiently deaminate nucleotides at positions 3-11 according to the numbering scheme in FIG. 3. In view of the results provided herein regarding the nucleotides that can be targeted by Cas9:deaminase fusion proteins, a person of skill in the art will be able to design suitable guide RNAs to target the fusion proteins to a target sequence that comprises a nucleotide to be deaminated.

In some embodiments, the deaminase domain and the Cas9 domain are fused to each other via a linker. Various linker lengths and flexibilities between the deaminase domain (e.g., AID) and the Cas9 domain can be employed (e.g., ranging from very flexible linkers of the form (GGGGS)_(n) (SEQ ID NO: 5), (GGS)_(n), and (G)_(n) to more rigid linkers of the form (EAAAK)_(n) (SEQ ID NO: 6), (SGGS)_(n)(SEQ ID NO: 4288), SGSETPGTSESATPES (SEQ ID NO: 7) (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)_(n))³⁶ in order to achieve the optimal length for deaminase activity for the specific application. In some embodiments, the linker comprises a (GGS)_(n) motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (an SGSETPGTSESATPES (SEQ ID NO: 7) motif.

Some exemplary suitable nucleic-acid editing domains, e.g., deaminases and deaminase domains, that can be fused to Cas9 domains according to aspects of this disclosure are provided below. It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).

Human AID: (SEQ ID NO: 266) MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLR YISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGL RRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDL RDAFRTLGL (underline: nuclear localization sequence; double underline: nuclear export signal) Mouse AID: (SEQ ID NO: 267) MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELLFLR YISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGL RRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLPLYEVDDL RDAFRMLGF (underline: nuclear localization sequence; double underline: nuclear export signal) Dog AID: (SEQ ID NO: 268) MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLR YISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGL RRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVDDL RDAFRTLGL (underline: nuclear localization sequence; double underline: nuclear export signal) Bovine AID: (SEQ ID NO: 269) MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGCHVELLFLR YISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEG LRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDD LRDAFRTLGL (underline: nuclear localization sequence; double underline: nuclear export signal) Rat AID (SEQ ID NO: 5725) MAVGSKPKAALVGPHWERERIWCFLCSTGLGTQQTGQTSRWLRPAATQDPVSPPRSLLMKQRK FLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGYLRNKSGCHVELLFLRYISDWDLDPG RCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLTGWGALPAGLMSPARPSDYFYCW NTFVENHERTFKAWEGLHENSVRLSRRLRRILLPLYEVDDLRDAFRTLGL (underline: nuclear localization sequence; double underline: nuclear export signal) Mouse APOBEC-3: (SEQ ID NO: 270) MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGV FKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQIVRFLATHHNLSLDI FSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQ DSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKH LCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSP CPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTN FVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS (italic: nucleic acid editing domain) Rat APOBEC-3: (SEQ ID NO: 271) MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGV FKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQVLRFLATHHNLSLDI FSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQ DSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYNQRVKH LCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVIITCYLTWSP CPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTN FVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDFGNLQLGPPMS (italic: nucleic acid editing domain) Rhesus macaque APOBEC-3G: (SEQ ID NO: 272) MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKYHPEM RF LRWFHKWRQLHHDQEYKVTWYVSWSPCTRCANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQ ALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHL MDPGTFTSNFNNKPWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGRHAE LCFLDLIPFWKLDGQQYRVTCFTSWSPCFSCAQEMAKFISNNEHVSLCIFAARIYDDQGRYQE GLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI (italic: nucleic acid editing domain; underline: cytoplasmic localization signal) Chimpanzee APOBEC-3G: (SEQ ID NO: 273) MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSKLK YHPEMRFFHWFSKWRKLHRDQEYEVIWYISWSPCTKCTRDVATFLAEDPKVTLTIFVARLYYF WDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIML GEILRHSMDPPTFTSNFNNELWVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPHKHGF LEGRHAELCFLDVIPFWKLDLHQDYRVTCFTSWSPCFSCAQEMAKFISNNKHVSLCIFAARIY DDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLEEHSQALSGRLRAI LQNQGN (italic: nucleic acid editing domain; underline: cytoplasmic localization signal) Green monkey APOBEC-3G: (SEQ ID NO: 274) MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYPEAK DHPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRCANSVATFLAEDPKVTLTIFVARLYYF WKPDYQQALRILCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATL GELLRHVMDPGTFTSNFNNKPWVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPDRHGF PKGRHAELCFLDLIPFWKLDDQQYRVTCFTSWSPCFSCAQKMAKFISNNKHVSLCIFAARIYD DQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI (italic: nucleic acid editing domain; underline: cytoplasmic localization signal) Human APOBEC-3G: (SEQ ID NO: 275) MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELK YHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYF WDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIML GEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGF LEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIY DDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAI LQNQEN (italic: nucleic acid editing domain; underline: cytoplasmic localization signal) Human APOBEC-3F: (SEQ ID NO: 276) MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQ VYSQPEHHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTL TISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPW YKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVK HHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECA GEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCW ENFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE (italic: nucleic acid editing domain) Human APOBEC-3B: (SEQ ID NO: 277) MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFR GQVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPN VTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQF MPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLD NGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWS PCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTY DEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (italic: nucleic acid editing domain) Rat APOBEC-3B: (SEQ ID NO: 5729) MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKN NFLCYEVNGMDCALPVPLRQGVFRKQGHIHAELCFIYWFHDKVLRVLSPMEEFKVT WYMSWSPCSKCAEQVARFLAAHRNLSLAIFSSRLYYYLRNPNYQQKLCRLIQEGVH VAAMDLPEFKKCWNKFVDNDGQPFRPWMRLRINFSFYDCKLQEIFSRMNLLREDVF YLQFNNSHRVKPVQNRYYRRKSYLCYQLERANGQEPLKGYLLYKKGEQHVEILFLE KMRSMELSQVRITCYLTWSPCPNCARQLAAFKKDHPDLILRIYTSRLYFYWRKKFQK GLCTLWRSGIHVDVMDLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKE SWGL Bovine APOBEC-3B: (SEQ ID NO: 5730) DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVL FKQQFGNQPRVPAPYYRRKTYLCYQLKQRNDLTLDRGCFRNKKQRHAEIRFIDKINS LDLNPSQSYKIICYITWSPCPNCANELVNFITRNNHLKLEIFASRLYFHWIKSFKMGLQ DLQNAGISVAVMTHTEFEDCWEQFVDNQSRPFQPWDKLEQYSASIRRRLQRILTAPI Chimpanzee APOBEC -3B: (SEQ ID NO: 5731) MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFR GQMYSQPEHHAEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHP NVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYNEGQPF MPWYKFDDNYAFLHRTLKEIIRHLMDPDTFTFNFNNDPLVLRRHQTYLCYEVERLD NGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFIS WSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIM TYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPP PPQSPGPCLPLCSEPPLGSLLPTGRPAPSLPFLLTASFSFPPPASLPPLPSLSLSPGHLPVP SFHSLTSCSIQPPCSSRIRETEGWASVSKEGRDLG Human APOBEC-3C: (SEQ ID NO: 278) MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVF RNQVDSETHCHAERCFLSWFCDDILSPNTKYQVIWYTSWSPCPDCAGEVAEFLARHSN VNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKP WKGLKTNFRLLKRRLRESLQ (italic: nucleic acid editing domain) Gorilla APOB EC3C (SEQ ID NO: 5726) MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVF RNQVDSETHCHAERCFLSWFCDDILSPNTNYQVIWYTSWSPCPECAGEVAEFLARHSN VNLTIFTARLYYFQDTDYQEGLRSLSQEGVAVKIMDYKDFKYCWENFVYNDDEPFK PWKGLKYNFRFLKRRLQEILE (italic: nucleic acid editing domain) Human APOBEC-3A: (SEQ ID NO: 279) MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLH NQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQ ENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGC PFQPWDGLDEHSQALSGRLRAILQNQGN (italic: nucleic acid editing domain) Rhesus macaque APOBEC-3A: (SEQ ID NO: 5727) MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVPMDERR GFLCNKAKNVPCGDYGCHVELRFLCEVPSWQLDPAQTYRVTWFISWSPCFRRGCAGQ VRVFLQENKHVRLRIFAARIYDYDPLYQEALRTLRDAGAQVSIMTYEEFKHCWDTF VDRQGRPFQPWDGLDEHSQALSGRLRAILQNQGN (italic: nucleic acid editing domain) Bovine APOBEC-3A: (SEQ ID NO: 5728) MDEYTFTENFNNQGWPSKTYLCYEMERLDGDATIPLDEYKGFVRNKGLDQPEKPCH AELYFLGKIHSWNLDRNQHYRLTCFISWSPCYDCAQKLTTFLKENHHISLHILASRIYTH NRFGCHQSGLCELQAAGARITIMTFEDFKHCWETFVDHKGKPFQPWEGLNVKSQAL CTELQAILKTQQN (italic: nucleic acid editing domain) Human APOBEC-3H: (SEQ ID NO: 280) MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKCHAEI CFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLGIFASRLYYHWC KPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRA IKRRLERIKIPGVRAQGRYMDILCDAEV (italic: nucleic acid editing domain) Rhesus macaque APOBEC-3H: (SEQ ID NO: 5732) MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAE IRFINKIKSMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYH WRPNYQEGLLLLCGSQVPVEVMGLPEFTDCWENFVDHKEPPSFNPSEKLEELDKNS QAIKRRLERIKSRSVDVLENGLRSLQLGPVTPSSSIRNSR Human APOBEC-3D: (SEQ ID NO: 281) MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFR GPVLPKRQSNHRQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPCVV KVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCW ENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKACG RNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNY EVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGAS VKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ (italic: nucleic acid editing domain) Human APOBEC-1: (SEQ ID NO: 282) MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKN TTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYV ARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYP PLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIH PSVAWR Mouse APOBEC-1: (SEQ ID NO: 283) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQN TSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIA RLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHL WVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK Rat APOBEC-1: (SEQ ID NO: 284) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW VRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK Human APOBEC-2: (SEQ ID NO: 5733) MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRN VEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALR YNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAG CKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK Mouse APOBEC-2: (SEQ ID NO: 5734) MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFR NVEYSSGRNKTFLCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDP ALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLK EAGCKLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK Rat APOBEC-2: (SEQ ID NO: 5735) MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFR NVEYSSGRNKTFLCYVVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDP ALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLK EAGCKLRIMKPQDFEYLWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK Bovine APOBEC-2: (SEQ ID NO: 5736) MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRN VEYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALR YMVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEA GCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK Petromyzon marinus CDA1 (pmCDA1) (SEQ ID NO: 5738) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNK PQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRG NGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQ LNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV Human APOBEC3G D316R_D317R (SEQ ID NO: 5739) MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQ VYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDP KVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQ RELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVER MHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTC FTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMT YSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN Human APOBEC3G chain A (SEQ ID NO: 5740) MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI FTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLD EHSQDLSGRLRAILQ Human APOBEC3G chain A D120R_D121R (SEQ ID NO: 5741) MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI FTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDE HSQDLSGRLRAILQ

In some embodiments, fusion proteins as provided herein comprise the full-length amino acid of a nucleic acid editing enzyme, e.g., one of the sequences provided above. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length sequence of a nucleic acid editing enzyme, but only a fragment thereof. For example, in some embodiments, a fusion protein provided herein comprises a Cas9 domain and a fragment of a nucleic acid editing enzyme, e.g., wherein the fragment comprises a nucleic acid editing domain. Exemplary amino acid sequences of nucleic acid editing domains are shown in the sequences above as italicized letters, and additional suitable sequences of such domains will be apparent to those of skill in the art.

Additional suitable nucleic-acid editing enzyme sequences, e.g., deaminase enzyme and domain sequences, that can be used according to aspects of this invention, e.g., that can be fused to a nuclease-inactive Cas9 domain, will be apparent to those of skill in the art based on this disclosure. In some embodiments, such additional enzyme sequences include deaminase enzyme or deaminase domain sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar to the sequences provided herein. Additional suitable Cas9 domains, variants, and sequences will also be apparent to those of skill in the art. Examples of such additional suitable Cas9 domains include, but are not limited to, D10A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838 the entire contents of which are incorporated herein by reference). In some embodiments, the Cas9 comprises a histidine residue at position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. The presence of the catalytic residue H840 restores the acvitity of the Cas9 to cleave the non-edited strand containing a G opposite the targeted C. Restoration of H840 does not result in the cleavage of the target strand containing the C.

Additional suitable strategies for generating fusion proteins comprising a Cas9 domain and a deaminase domain will be apparent to those of skill in the art based on this disclosure in combination with the general knowledge in the art. Suitable strategies for generating fusion proteins according to aspects of this disclosure using linkers or without the use of linkers will also be apparent to those of skill in the art in view of the instant disclosure and the knowledge in the art. For example, Gilbert et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013; 154(2):442-51, showed that C-terminal fusions of Cas9 with VP64 using 2 NLS's as a linker (SPKKKRKVEAS, SEQ ID NO: 599), can be employed for transcriptional activation. Mali et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013; 31(9):833-8, reported that C-terminal fusions with VP64 without linker can be employed for transcriptional activation. And Maeder et al., CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013; 10: 977-979, reported that C-terminal fusions with VP64 using a Gly4Ser (SEQ ID NO: 5) linker can be used as transcriptional activators. Recently, dCas9-FokI nuclease fusions have successfully been generated and exhibit improved enzymatic specificity as compared to the parental Cas9 enzyme (In Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82, and in Tsai S Q, Wyvekens N, Khayter C, Foden J A, Thapar V, Reyon D, Goodwin M J, Aryee M J, Joung J K. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014; 32(6):569-76. PMID: 24770325 a SGSETPGTSESATPES (SEQ ID NO: 7) or a GGGGS (SEQ ID NO: 5) linker was used in FokI-dCas9 fusion proteins, respectively).

Some aspects of this disclosure provide fusion proteins comprising (i) a Cas9 enzyme or domain (e.g., a first protein); and (ii) a nucleic acid-editing enzyme or domain (e.g., a second protein). In some aspects, the fusion proteins provided herein further include (iii) a programmable DNA-binding protein, for example, a zinc-finger domain, a TALE, or a second Cas9 protein (e.g., a third protein). Without wishing to be bound by any particular theory, fusing a programmable DNA-binding protein (e.g., a second Cas9 protein) to a fusion protein comprising (i) a Cas9 enzyme or domain (e.g., a first protein); and (ii) a nucleic acid-editing enzyme or domain (e.g., a second protein) may be useful for improving specificity of the fusion protein to a target nucleic acid sequence, or for improving specificity or binding affinity of the fusion protein to bind target nucleic acid sequence that does not contain a canonical PAM (NGG) sequence. In some embodiments, the third protein is a Cas9 protein (e.g, a second Cas9 protein). In some embodiments, the third protein is any of the Cas9 proteins provided herein. In some embodiments, the third protein is fused to the fusion protein N-terminal to the Cas9 protein (e.g., the first protein). In some embodiments, the third protein is fused to the fusion protein C-terminal to the Cas9 protein (e.g., the first protein). In some embodiments, the Cas9 domain (e.g., the first protein) and the third protein (e.g., a second Cas9 protein) are fused via a linker (e.g., a second linker). In some embodiments, the linker comprises a (GGGGS)_(n) (SEQ ID NO: 5), a (G)_(n), an (EAAAK)_(n) (SEQ ID NO: 6), a (GGS)_(n), (SGGS)_(n) (SEQ ID NO: 4288), an SGSETPGTSESATPES (SEQ ID NO: 7), or an (XP)_(n) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. In some embodiments, the general architecture of exemplary Cas9 fusion proteins provided herein comprises the structure:

-   -   [NH2]-[nucleic acid-editing enzyme or domain]-[Cas9]-[third         protein]-[COOH];     -   [NH2]-[third protein]-[Cas9]-[nucleic acid-editing enzyme or         domain]-[COOH];     -   [NH2]-[Cas9]-[nucleic acid-editing enzyme or domain]-[third         protein]-[COOH];     -   [NH2]-[third protein]-[nucleic acid-editing enzyme or         domain]-[Cas9]-[COOH];     -   [NH2]-[UGI]-[nucleic acid-editing enzyme or         domain]-[Cas9]-[third protein]-[COOH];     -   [NH2]-[UGI]-[third protein]-[Cas9]-[nucleic acid-editing enzyme         or domain]-[COOH];     -   [NH2]-[UGI]-[Cas9]-[nucleic acid-editing enzyme or         domain]-[third protein]-[COOH];     -   [NH2]-[UGI]-[third protein]-[nucleic acid-editing enzyme or         domain]-[Cas9]-[COOH];     -   [NH2]-[nucleic acid-editing enzyme or domain]-[Cas9]-[third         protein]-[UGI]-[COOH];     -   [NH2]-[third protein]-[Cas9]-[nucleic acid-editing enzyme or         domain]-[UGI]-[COOH];     -   [NH2]-[Cas9]-[nucleic acid-editing enzyme or domain]-[third         protein]-[UGI]-[COOH]; or     -   [NH2]-[third protein]-[nucleic acid-editing enzyme or         domain]-[Cas9]-[UGI]-[COOH];         wherein NH2 is the N-terminus of the fusion protein, and COOH is         the C-terminus of the fusion protein. In some embodiments, the         “]-[” used in the general architecture above indicates the         presence of an optional linker sequence. In other examples, the         general architecture of exemplary Cas9 fusion proteins provided         herein comprises the structure:     -   [NH2]-[nucleic acid-editing enzyme or domain]-[Cas9]-[second         Cas9 protein]-[COOH];     -   [NH2]-[second Cas9 protein]-[Cas9]-[nucleic acid-editing enzyme         or domain]-[COOH];     -   [NH2]-[Cas9]-[nucleic acid-editing enzyme or domain]-[second         Cas9 protein]-[COOH];     -   [NH2]-[second Cas9 protein]-[nucleic acid-editing enzyme or         domain]-[Cas9]-[COOH];     -   [NH2]-[UGI]-[nucleic acid-editing enzyme or         domain]-[Cas9]-[second Cas9 protein]-[COOH], [NH2]-[UGI]-[second         Cas9 protein]-[Cas9]-[nucleic acid-editing enzyme or         domain]-[COOH];     -   [NH2]-[UGI]-[Cas9]-[nucleic acid-editing enzyme or         domain]-[second Cas9 protein]-[COOH];     -   [NH2]-[UGI]-[second Cas9 protein]-[nucleic acid-editing enzyme         or domain]-[Cas9]-[COOH];     -   [NH2]-[nucleic acid-editing enzyme or domain]-[Cas9]-[second         Cas9 protein]-[UGI]-[COOH];     -   [NH2]-[second Cas9 protein]-[Cas9]-[nucleic acid-editing enzyme         or domain]-[UGI]-[COOH];     -   [NH2]-[Cas9]-[nucleic acid-editing enzyme or domain]-[second         Cas9 protein]-[UGI]-[COOH]; or     -   [NH2]-[second Cas9 protein]-[nucleic acid-editing enzyme or         domain]-[Cas9]-[UGI]-[COOH];         wherein NH₂ is the N-terminus of the fusion protein, and COOH is         the C-terminus of the fusion protein. In some embodiments, the         “]-[” used in the general architecture above indicates the         presence of an optional linker sequence. In some embodiments,         the second Cas9 is a dCas9 protein. In some examples, the         general architecture of exemplary Cas9 fusion proteins provided         herein comprises a structure as shown in FIG. 3. It should be         appreciated that any of the proteins provided in any of the         general architectures of exemplary Cas9 fusion proteins may be         connected by one or more of the linkers provided herein. In some         embodiments, the linkers are the same. In some embodiments, the         linkers are different. In some embodiments, one or more of the         proteins provided in any of the general architectures of         exemplary Cas9 fusion proteins are not fused via a linker. In         some embodiments, the fusion proteins further comprise a nuclear         targeting sequence, for example a nuclear localization sequence.         In some embodiments, fusion proteins provided herein further         comprise a nuclear localization sequence (NLS). In some         embodiments, the NLS is fused to the N-terminus of the fusion         protein. In some embodiments, the NLS is fused to the C-terminus         of the fusion protein. In some embodiments, the NLS is fused to         the N-terminus of the third protein. In some embodiments, the         NLS is fused to the C-terminus of the third protein. In some         embodiments, the NLS is fused to the N-terminus of the Cas9         protein. In some embodiments, the NLS is fused to the C-terminus         of the Cas9 protein. In some embodiments, the NLS is fused to         the N-terminus of the nucleic acid-editing enzyme or domain. In         some embodiments, the NLS is fused to the C-terminus of the         nucleic acid-editing enzyme or domain. In some embodiments, the         NLS is fused to the N-terminus of the UGI protein. In some         embodiments, the NLS is fused to the C-terminus of the UGI         protein. In some embodiments, the NLS is fused to the fusion         protein via one or more linkers. In some embodiments, the NLS is         fused to the fusion protein without a linker

Uracil Glycosylase Inhibitor Fusion Proteins

Some aspects of the disclosure relate to fusion proteins that comprise a uracil glycosylase inhibitor (UGI) domain. In some embodiments, any of the fusion proteins provided herein that comprise a Cas9 domain (e.g., a nuclease active Cas9 domain, a nuclease inactive dCas9 domain, or a Cas9 nickase) may be further fused to a UGI domain either directly or via a linker. Some aspects of this disclosure provide deaminase-dCas9 fusion proteins, deaminase-nuclease active Cas9 fusion proteins and deaminase-Cas9 nickase fusion proteins with increased nucleobase editing efficiency. Without wishing to be bound by any particular theory, cellular DNA-repair response to the presence of U:G heteroduplex DNA may be responsible for the decrease in nucleobase editing efficiency in cells. For example, uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells, which may initiate base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. As demonstrated in the Examples below, Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity. Thus, this disclosure contemplates a fusion protein comprising dCas9-nucleic acid editing domain further fused to a UGI domain. This disclosure also contemplates a fusion protein comprising a Cas9 nickase-nucleic acid editing domain further fused to a UGI domain. It should be understood that the use of a UGI domain may increase the editing efficiency of a nucleic acid editing domain that is capable of catalyzing a C to U change. For example, fusion proteins comprising a UGI domain may be more efficient in deaminating C residues. In some embodiments, the fusion protein comprises the structure:

-   -   [deaminase]-[optional linker sequence]-[dCas9]-[optional linker         sequence]-[UGI];     -   [deaminase]-[optional linker sequence]-[UGI]-[optional linker         sequence]-[dCas9];     -   [UGI]-[optional linker sequence]-[deaminase]-[optional linker         sequence]-[dCas9];     -   [UGI]-[optional linker sequence]-[dCas9]-[optional linker         sequence]-[deaminase];     -   [dCas9]-[optional linker sequence]-[deaminase]-[optional linker         sequence]-[UGI]; or     -   [dCas9]-[optional linker sequence]-[UGI]-[optional linker         sequence]-[deaminase].         In other embodiments, the fusion protein comprises the         structure:     -   [deaminase]-[optional linker sequence]-[Cas9 nickase]-[optional         linker sequence]-[UGI];     -   [deaminase]-[optional linker sequence]-[UGI]-[optional linker         sequence]-[Cas9 nickase];     -   [UGI]-[optional linker sequence]-[deaminase]-[optional linker         sequence]-[Cas9 nickase];     -   [UGI]-[optional linker sequence]-[Cas9 nickase]-[optional linker         sequence]-[deaminase];     -   [Cas9 nickase]-[optional linker sequence]-[deaminase]-[optional         linker sequence]-[UGI]; or     -   [Cas9 nickase]-[optional linker sequence]-[UGI]-[optional linker         sequence]-[deaminase].

In some embodiments, the fusion proteins provided herein do not comprise a linker sequence. In some embodiments, one or both of the optional linker sequences are present.

In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker sequence. In some embodiments, the fusion proteins comprising a UGI further comprise a nuclear targeting sequence, for example a nuclear localization sequence. In some embodiments, fusion proteins provided herein further comprise a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the UGI protein. In some embodiments, the NLS is fused to the C-terminus of the UGI protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the C-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the N-terminus of the second Cas9. In some embodiments, the NLS is fused to the C-terminus of the second Cas9. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. In some embodiments, the NLS comprises an amino acid sequence as set forth in SEQ ID NO: 741 or SEQ ID NO: 742.

In some embodiments, a UGI domain comprises a wild-type UGI or a UGI as set forth in SEQ ID NO: 600. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 600. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in SEQ ID NO: 600. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 600 or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 600. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as “UGI variants.” A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 600. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the corresponding fragment of wild-type UGI or a UGI as set forth in SEQ ID NO: 600. In some embodiments, the UGI comprises the following amino acid sequence: >sp|P14739|UNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 600)

Suitable UGI protein and nucleotide sequences are provided herein and additional suitable UGI sequences are known to those in the art, and include, for example, those published in Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J. Biol. Chem. 264:1163-1171(1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J. Biol. Chem. 272:21408-21419(1997); Ravishankar et al., X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG. Nucleic Acids Res. 26:4880-4887(1998); and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase. J. Mol. Biol. 287:331-346(1999), the entire contents of each are incorporated herein by reference.

It should be appreciated that additional proteins may be uracil glycosylase inhibitors. For example, other proteins that are capable of inhibiting (e.g., sterically blocking) a uracil-DNA glycosylase base-excision repair enzyme are within the scope of this disclosure. Additionally, any proteins that block or inhibit base-excision repair as also within the scope of this disclosure. In some embodiments, a protein that binds DNA is used. In another embodiment, a substitute for UGI is used. In some embodiments, a uracil glycosylase inhibitor is a protein that binds single-stranded DNA. For example, a uracil glycosylase inhibitor may be a Erwinia tasmaniensis single-stranded binding protein. In some embodiments, the single-stranded binding protein comprises the amino acid sequence (SEQ ID NO: 322). In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil. In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil in DNA. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein that does not excise uracil from the DNA. For example, a uracil glycosylase inhibitor is a UdgX. In some embodiments, the UdgX comprises the amino acid sequence (SEQ ID NO: 323). As another example, a uracil glycosylase inhibitor is a catalytically inactive UDG. In some embodiments, a catalytically inactive UDG comprises the amino acid sequence (SEQ ID NO: 324). It should be appreciated that other uracil glycosylase inhibitors would be apparent to the skilled artisan and are within the scope of this disclosure. In some embodiments, a uracil glycosylase inhibitor is a protein that is homologous to any one of SEQ ID NOs: 322-324. In some embodiments, a uracil glycosylase inhibitor is a protein that is at least 50% identical, at least 55% identical at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to any one of SEQ ID NOs: 322-324.

Erwinia tasmaniensis SSB (themostable single-stranded DNA binding protein) (SEQ ID NO: 322) MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKQTGETK EKTEWHRVVLFGKLAEVAGEYLRKGSQVYIEGALQTRKWTDQAGVEKYTT EVVVNVGGTMQMLGGRSQGGGASAGGQNGGSNNGWGQPQQPQGGNQFSGG AQQQARPQQQPQQNNAPANNEPPIDFDDDIP UdgX (binds to Uracil in DNA but does not excise) (SEQ ID NO: 323) MAGAQDFVPHTADLAELAAAAGECRGCGLYRDATQAVFGAGGRSARIMMI GEQPGDKEDLAGLPFVGPAGRLLDRALEAADIDRDALYVTNAVKHFKFTR AAGGKRRIHKTPSRTEVVACRPWLIAEMTSVEPDVVVLLGATAAKALLGN DFRVTQHRGEVLHVDDVPGDPALVATVHPSSLLRGPKEERESAFAGLVDD LRVAADVRP UDG (catalytically inactive human UDG, binds to Uracil in DNA but does not excise) (SEQ ID NO: 324) MIGQKTLYSFFSPSPARKRHAPSPEPAVQGTGVAGVPEESGDAAAIPAKK APAGQEEPGTPPSSPLSAEQLDRIQRNKAAALLRLAARNVPVGFGESWKK HLSGEFGKPYFIKLMGFVAEERKHYTVYPPPHQVFTWTQMCDIKDVKVVI LGQEPYHGPNQAHGLCFSVQRPVPPPPSLENIYKELSTDIEDFVHPGHGD LSGWAKQGVLLLNAVLTVRAHQANSHKERGWEQFTDAVVSWLNQNSNGLV FLLWGSYAQKKGSAIDRKRHHVLQTAHPSPLSVYRGFFGCRHFSKTNELL QKSGKKPIDWKEL

In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a rat APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is a human APOBEC1 (SEQ ID No: 284). In some embodiments, the deaminase is a Petromyzon marinus cytidine deaminase 1 (pmCDA1). In some embodiments, the deaminase is a human APOBEC3G (SEQ ID NO: 275). In some embodiments, the deaminase is a fragment of the human APOBEC3G (SEQ ID NO: 5740). In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R_D317R mutation (SEQ ID NO: 5739). In some embodiments, the deaminase is a frantment of the human APOBEC3G and comprising mutations corresponding to the D316R_D317R mutations in SEQ ID NO: 275 (SEQ ID NO: 5741).

In some embodiments, the linker comprises a (GGGS)_(n) (SEQ ID NO: 265), (GGGGS)_(n) (SEQ ID NO: 5), a (G)_(n), an (EAAAK)_(n) (SEQ ID NO: 6), a (GGS)_(n), an SGSETPGTSESATPES (SEQ ID NO: 7), or an (XP)_(n) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30.

Suitable UGI protein and nucleotide sequences are provided herein and additional suitable UGI sequences are known to those in the art, and include, for example, those published in Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J. Biol. Chem. 264:1163-1171(1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J. Biol. Chem. 272:21408-21419(1997); Ravishankar et al., X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG. Nucleic Acids Res. 26:4880-4887(1998); and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase. J. Mol. Biol. 287:331-346(1999), the entire contents of which are incorporated herein by reference. In some embodiments, the optional linker comprises a (GGS)_(n) motif, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the optional linker comprises a (GGS)_(n) motif, wherein n is 1, 3, or 7. In some embodiments, the optional linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7), which is also referred to as the XTEN linker in the Examples.

In some embodiments, a Cas9 nickase may further facilitate the removal of a base on the non-edited strand in an organism whose genome is edited in vivo. The Cas9 nickase, as described herein, may comprise a D10A mutation in SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260. In some embodiments, the Cas9 nickase of this disclosure may comprise a histidine at mutation 840 of SEQ ID NO: 10, or a corresponding residue in any of SEQ ID NOs: 11-260. Such fusion proteins comprising the Cas9 nickase, can cleave a single strand of the target DNA sequence, e.g., the strand that is not being edited. Without wishing to be bound by any particular theory, this cleavage may inhibit mis-match repair mechanisms that reverse a C to U edit made by the deaminase.

Cas9 Complexes with Guide RNAs

Some aspects of this disclosure provide complexes comprising any of the fusion proteins provided herein, and a guide RNA bound to a Cas9 domain (e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase) of fusion protein.

In some embodiments, the guide RNA is from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of a mammal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the guide RNA is complementary to a sequence associated with a disease or disorder. In some embodiments, the guide RNA is complementary to a sequence associated with a disease or disorder having a mutation in a gene selected from the genes disclosed in any one of Tables 1-3. In some embodiments, the guide RNA comprises a nucleotide sequence of any one of the guide sequences provided in Table 2 or Table 3. Exemplary sequences in the human genome that may be targeted by the complexes of this disclosure are provided herein in Tables 1-3.

Methods of Using Cas9 Fusion Proteins

Some aspects of this disclosure provide methods of using the Cas9 proteins, fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule (a) with any of the Cas9 proteins or fusion proteins provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence; or (b) with a Cas9 protein, a Cas9 fusion protein, or a Cas9 protein or fusion protein complex with at least one gRNA as provided herein. In some embodiments, the 3′ end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

In some embodiments, the target DNA sequence comprises a sequence associated with a disease or disorder. In some embodiments, the target DNA sequence comprises a point mutation associated with a disease or disorder. In some embodiments, the activity of the Cas9 protein, the Cas9 fusion protein, or the complex results in a correction of the point mutation. In some embodiments, the target DNA sequence comprises a T→C point mutation associated with a disease or disorder, and wherein the deamination of the mutant C base results in a sequence that is not associated with a disease or disorder. In some embodiments, the target DNA sequence encodes a protein and wherein the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon. In some embodiments, the deamination of the mutant C results in a change of the amino acid encoded by the mutant codon. In some embodiments, the deamination of the mutant C results in the codon encoding the wild-type amino acid. In some embodiments, the contacting is in vivo in a subject. In some embodiments, the subject has or has been diagnosed with a disease or disorder. In some embodiments, the disease or disorder is cystic fibrosis, phenylketonuria, epidermolytic hyperkeratosis (EHK), Charcot-Marie-Toot disease type 4J, neuroblastoma (NB), von Willebrand disease (vWD), myotonia congenital, hereditary renal amyloidosis, dilated cardiomyopathy (DCM), hereditary lymphedema, familial Alzheimer's disease, HIV, Prion disease, chronic infantile neurologic cutaneous articular syndrome (CINCA), desmin-related myopathy (DRM), a neoplastic disease associated with a mutant PI3KCA protein, a mutant CTNNB1 protein, a mutant HRAS protein, or a mutant p53 protein.

Some embodiments provide methods for using the Cas9 DNA editing fusion proteins provided herein. In some embodiments, the fusion protein is used to introduce a point mutation into a nucleic acid by deaminating a target nucleobase, e.g., a C residue. In some embodiments, the deamination of the target nucleobase results in the correction of a genetic defect, e.g., in the correction of a point mutation that leads to a loss of function in a gene product. In some embodiments, the genetic defect is associated with a disease or disorder, e.g., a lysosomal storage disorder or a metabolic disease, such as, for example, type I diabetes. In some embodiments, the methods provided herein are used to introduce a deactivating point mutation into a gene or allele that encodes a gene product that is associated with a disease or disorder. For example, in some embodiments, methods are provided herein that employ a Cas9 DNA editing fusion protein to introduce a deactivating point mutation into an oncogene (e.g., in the treatment of a proliferative disease). A deactivating mutation may, in some embodiments, generate a premature stop codon in a coding sequence, which results in the expression of a truncated gene product, e.g., a truncated protein lacking the function of the full-length protein.

In some embodiments, the purpose of the methods provide herein is to restore the function of a dysfunctional gene via genome editing. The Cas9 deaminase fusion proteins provided herein can be validated for gene editing-based human therapeutics in vitro, e.g., by correcting a disease-associated mutation in human cell culture. It will be understood by the skilled artisan that the fusion proteins provided herein, e.g., the fusion proteins comprising a Cas9 domain and a nucleic acid deaminase domain can be used to correct any single point T->C or A->G mutation. In the first case, deamination of the mutant C back to U corrects the mutation, and in the latter case, deamination of the C that is base-paired with the mutant G, followed by a round of replication, corrects the mutation.

An exemplary disease-relevant mutation that can be corrected by the provided fusion proteins in vitro or in vivo is the H1047R (A3140G) polymorphism in the PI3KCA protein. The phosphoinositide-3-kinase, catalytic alpha subunit (PI3KCA) protein acts to phosphorylate the 3-OH group of the inositol ring of phosphatidylinositol. The PI3KCA gene has been found to be mutated in many different carcinomas, and thus it is considered to be a potent oncogene.³⁷ In fact, the A3140G mutation is present in several NCI-60 cancer cell lines, such as, for example, the HCT116, SKOV3, and T47D cell lines, which are readily available from the American Type Culture Collection (ATCC).³⁸

In some embodiments, a cell carrying a mutation to be corrected, e.g., a cell carrying a point mutation, e.g., an A3140G point mutation in exon 20 of the PI3KCA gene, resulting in a H1047R substitution in the PI3KCA protein, is contacted with an expression construct encoding a Cas9 deaminase fusion protein and an appropriately designed sgRNA targeting the fusion protein to the respective mutation site in the encoding PI3KCA gene. Control experiments can be performed where the sgRNAs are designed to target the fusion enzymes to non-C residues that are within the PI3KCA gene. Genomic DNA of the treated cells can be extracted, and the relevant sequence of the PI3KCA genes PCR amplified and sequenced to assess the activities of the fusion proteins in human cell culture.

It will be understood that the example of correcting point mutations in PI3KCA is provided for illustration purposes and is not meant to limit the instant disclosure. The skilled artisan will understand that the instantly disclosed DNA-editing fusion proteins can be used to correct other point mutations and mutations associated with other cancers and with diseases other than cancer including other proliferative diseases.

The successful correction of point mutations in disease-associated genes and alleles opens up new strategies for gene correction with applications in therapeutics and basic research. Site-specific single-base modification systems like the disclosed fusions of Cas9 and deaminase enzymes or domains also have applications in “reverse” gene therapy, where certain gene functions are purposely suppressed or abolished. In these cases, site-specifically mutating Trp (TGG), Gln (CAA and CAG), or Arg (CGA) residues to premature stop codons (TAA, TAG, TGA) can be used to abolish protein function in vitro, ex vivo, or in vivo.

The instant disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation that can be corrected by a Cas9 DNA editing fusion protein provided herein. For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a PI3KCA point mutation as described above, an effective amount of a Cas9 deaminase fusion protein that corrects the point mutation or introduces a deactivating mutation into the disease-associated gene. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect.

The instant disclosure provides methods for the treatment of additional diseases or disorders, e.g., diseases or disorders that are associated or caused by a point mutation that can be corrected by deaminase-mediated gene editing. Some such diseases are described herein, and additional suitable diseases that can be treated with the strategies and fusion proteins provided herein will be apparent to those of skill in the art based on the instant disclosure. Exemplary suitable diseases and disorders are listed below. It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Exemplary suitable diseases and disorders include, without limitation, cystic fibrosis (see, e.g., Schwank et al., Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell. 2013; 13: 653-658; and Wu et. al., Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell. 2013; 13: 659-662, neither of which uses a deaminase fusion protein to correct the genetic defect); phenylketonuria—e.g., phenylalanine to serine mutation at position 835 (mouse) or 240 (human) or a homologous residue in phenylalanine hydroxylase gene (T>C mutation)—see, e.g., McDonald et al., Genomics. 1997; 39:402-405; Bernard-Soulier syndrome (BSS)—e.g., phenylalanine to serine mutation at position 55 or a homologous residue, or cysteine to arginine at residue 24 or a homologous residue in the platelet membrane glycoprotein IX (T>C mutation)—see, e.g., Noris et al., British Journal of Haematology. 1997; 97: 312-320, and Ali et al., Hematol. 2014; 93: 381-384; epidermolytic hyperkeratosis (EHK)—e.g., leucine to proline mutation at position 160 or 161 (if counting the initiator methionine) or a homologous residue in keratin 1 (T>C mutation)—see, e.g., Chipev et al., Cell. 1992; 70: 821-828, see also accession number P04264 in the UNIPROT database at www[dot]uniprot[dot]org; chronic obstructive pulmonary disease (COPD)—e.g., leucine to proline mutation at position 54 or 55 (if counting the initiator methionine) or a homologous residue in the processed form of α₁-antitrypsin or residue 78 in the unprocessed form or a homologous residue (T>C mutation)—see, e.g., Poller et al., Genomics. 1993; 17: 740-743, see also accession number P01011 in the UNIPROT database; Charcot-Marie-Toot disease type 4J—e.g., isoleucine to threonine mutation at position 41 or a homologous residue in FIG. 4 (T>C mutation)—see, e.g., Lenk et al., PLoS Genetics. 2011; 7: e1002104; neuroblastoma (NB)—e.g., leucine to proline mutation at position 197 or a homologous residue in Caspase-9 (T>C mutation)—see, e.g., Kundu et al., 3 Biotech. 2013, 3:225-234; von Willebrand disease (vWD)—e.g., cysteine to arginine mutation at position 509 or a homologous residue in the processed form of von Willebrand factor, or at position 1272 or a homologous residue in the unprocessed form of von Willebrand factor (T>C mutation)—see, e.g., Lavergne et al., Br. J. Haematol. 1992, see also accession number P04275 in the UNIPROT database; 82: 66-72; myotonia congenital—e.g., cysteine to arginine mutation at position 277 or a homologous residue in the muscle chloride channel gene CLCN1 (T>C mutation)—see, e.g., Weinberger et al., The J. of Physiology. 2012; 590: 3449-3464; hereditary renal amyloidosis—e.g., stop codon to arginine mutation at position 78 or a homologous residue in the processed form of apolipoprotein All or at position 101 or a homologous residue in the unprocessed form (T>C mutation)—see, e.g., Yazaki et al., Kidney Int. 2003; 64: 11-16; dilated cardiomyopathy (DCM)—e.g., tryptophan to Arginine mutation at position 148 or a homologous residue in the FOXD4 gene (T>C mutation), see, e.g., Minoretti et. al., Int. J. of Mol. Med. 2007; 19: 369-372; hereditary lymphedema—e.g., histidine to arginine mutation at position 1035 or a homologous residue in VEGFR3 tyrosine kinase (A>G mutation), see, e.g., Irrthum et al., Am. J. Hum. Genet. 2000; 67: 295-301; familial Alzheimer's disease—e.g., isoleucine to valine mutation at position 143 or a homologous residue in presenilin1 (A>G mutation), see, e.g., Gallo et. al., J. Alzheimer's disease. 2011; 25: 425-431; Prion disease—e.g., methionine to valine mutation at position 129 or a homologous residue in prion protein (A>G mutation)—see, e.g., Lewis et. al., J. of General Virology. 2006; 87: 2443-2449; chronic infantile neurologic cutaneous articular syndrome (CINCA)—e.g., Tyrosine to Cysteine mutation at position 570 or a homologous residue in cryopyrin (A>G mutation)—see, e.g., Fujisawa et. al. Blood. 2007; 109: 2903-2911; and desmin-related myopathy (DRM)—e.g., arginine to glycine mutation at position 120 or a homologous residue in αβ crystallin (A>G mutation)—see, e.g., Kumar et al., J. Biol. Chem. 1999; 274: 24137-24141. The entire contents of all references and database entries is incorporated herein by reference.

The instant disclosure provides lists of genes comprising pathogenic T>C or A>G mutations. Provided herein, are the names of these genes, their respective SEQ ID NOs, their gene IDs, and sequences flanking the mutation site. (Tables 2 and 3). In some instances, the gRNA sequences that can be used to correct the mutations in these genes are disclosed (Tables 2 and 3).

In some embodiments, a Cas9-deaminase fusion protein recognizes canonical PAMs and therefore can correct the pathogenic T>C or A>G mutations with canonical PAMs, e.g., NGG (listed in Tables 2 and 3, SEQ ID NOs: 2540-2702 and 5084-5260), respectively, in the flanking sequences. For example, the Cas9 proteins that recognize canonical PAMs comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 10, or to a fragment thereof comprising the RuvC and HNH domains of SEQ ID NO: 10.

It will be apparent to those of skill in the art that in order to target a Cas9:nucleic acid editing enzyme/domain fusion protein as disclosed herein to a target site, e.g., a site comprising a point mutation to be edited, it is typically necessary to co-express the Cas9:nucleic acid editing enzyme/domain fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein. In some embodiments, the guide RNA comprises a structure 5′-[guide sequence]-guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuu uuu-3′ (SEQ ID NO: 601), wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific target sequences are provided below.

Base Editor Efficiency

Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. An “indel”, as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels that is greater than 1:1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels may be determined using any suitable method, for example the methods used in the below Examples.

In some embodiments, the base editors provided herein are capable of limiting formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein are capable of limiting the formation of indels at a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%. The number of indels formed at a nucleic acid region may depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a base editor. In some embodiments, an number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a nucleic acid (e.g., a nucleic acid within the genome of a cell) to a base editor.

Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation, such as a point mutation, in a nucleic acid (e.g. a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, a intended mutation is a mutation that is generated by a specific base editor bound to a gRNA, specifically designed to generate the intended mutation. In some embodiments, the intended mutation is a mutation associated with a disease or disorder. In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a guanine (G) to adenine (A) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a guanine (G) to adenine (A) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a point mutation that generates a stop codon, for example, a premature stop codon within the coding region of a gene. In some embodiments, the intended mutation is a mutation that eliminates a stop codon. In some embodiments, the intended mutation is a mutation that alters the splicing of a gene. In some embodiments, the intended mutation is a mutation that alters the regulatory sequence of a gene (e.g., a gene promotor or gene repressor). In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations:unintended point mutations) that is greater than 1:1. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations:unintended point mutations) that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1, or more. It should be appreciated that the characteristics of the base editors described in the “Base Editor Efficiency” section, herein, may be applied to any of the fusion proteins, or methods of using the fusion proteins provided herein.

Methods for Editing Nucleic Acids

Some aspects of the disclosure provide methods for editing a nucleic acid. In some embodiments, the method is a method for editing a nucleobase of a nucleic acid (e.g., a base pair of a double-stranded DNA sequence). In some embodiments, the method comprises the steps of: a) contacting a target region of a nucleic acid (e.g., a double-stranded DNA sequence) with a complex comprising a base editor (e.g., a Cas9 domain fused to a cytidine deaminase domain) and a guide nucleic acid (e.g., gRNA), wherein the target region comprises a targeted nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, and d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase; and the method results in less than 20% indel formation in the nucleic acid. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, the first nucleobase is a cytosine. In some embodiments, the second nucleobase is a deaminated cytosine, or a uracil. In some embodiments, the third nucleobase is a guanine. In some embodiments, the fourth nucleobase is an adenine. In some embodiments, the first nucleobase is a cytosine, the second nucleobase is a deaminated cytosine, or a uracil, the third nucleobase is a guanine, and the fourth nucleobase is an adenine. In some embodiments, the method results in less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair (e.g., C:G->T:A). In some embodiments, the fifth nucleobase is a thymine. In some embodiments, at least 5% of the intended basepairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended basepairs are edited.

In some embodiments, the ratio of intended products to unintended products in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises a Cas9 domain. In some embodiments, the first base is cytosine, and the second base is not a G, C, A, or T. In some embodiments, the second base is uracil. In some embodiments, the first base is cytosine. In some embodiments, the second base is not a G, C, A, or T. In some embodiments, the second base is uracil. In some embodiments, the base editor inhibits base excision repair of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the base editor comprises UGI activity. In some embodiments, the base editor comprises nickase activity. In some embodiments, the intended edited basepair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair is within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a deamination window

In some embodiments, the disclosure provides methods for editing a nucleotide. In some embodiments, the disclosure provides a method for editing a nucleobase pair of a double-stranded DNA sequence. In some embodiments, the method comprises a) contacting a target region of the double-stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g., gRNA), where the target region comprises a target nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, d) cutting no more than one strand of said target region, wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase, and the second nucleobase is replaced with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited basepair, wherein the efficiency of generating the intended edited basepair is at least 5%. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, at least 5% of the intended basepairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended basepairs are edited. In some embodiments, the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended products at the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the first base is cytosine. In some embodiments, the second nucleobase is not G, C, A, or T. In some embodiments, the second base is uracil. In some embodiments, the base editor inhibits base excision repair of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the nucleobase editor comprises UGI activity. In some embodiments, the nucleobase edit comprises nickase activity. In some embodiments, the intended edited basepair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair occurs within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the nucleobase editor is any one of the base editors provided herein.

Kits, Vectors, Cells

Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a Cas9 protein or a Cas9 fusion protein as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the kit further comprises an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone.

Some aspects of this disclosure provide polynucleotides encoding a Cas9 protein of a fusion protein as provided herein. Some aspects of this disclosure provide vectors comprising such polynucleotides. In some embodiments, the vector comprises a heterologous promoter driving expression of polynucleotide.

Some aspects of this disclosure provide cells comprising a Cas9 protein, a fusion protein, a nucleic acid molecule encoding the fusion protein, a complex comprise the Cas9 protein and the gRNA, and/or a vector as provided herein.

The description of exemplary embodiments of the reporter systems above is provided for illustration purposes only and not meant to be limiting. Additional reporter systems, e.g., variations of the exemplary systems described in detail above, are also embraced by this disclosure.

EXAMPLES Example 1: Cas9 Deaminase Fusion Proteins

A number of Cas9:Deaminase fusion proteins were generated and deaminase activity of the generated fusions was characterized. The following deaminases were tested:

Human AID (hAID): (SEQ ID NO: 607) MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG NPYLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT FVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGLLD Human AID-DC (hAID-DC, truncated version of hAID with 7-fold increased activity): (SEQ ID NO: 608) MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT FVENHERTFKAWEGLHENSVRLSRQLRRILL Rat APOBEC1 (rAPOBEC1): (SEQ ID NO: 284) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ PQLTFFTIALQSCHYQRLPPHILWATGLK Human APOBEC1 (hAPOBEC1) (SEQ ID NO: 5724) MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKI WRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAI REFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYY HCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQ NHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR Petromyzon marinus (Lamprey) CDA1 (pmCDA1): (SEQ ID NO: 609) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFW GYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNV MVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL HTTKSPAV Human APOBEC3G (hAPOBEC3G): (SEQ ID NO: 610) MELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLA EDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQH CWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNE PWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAE LCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI FTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQ PWDGLDEHSQDLSGRLRAILQNQEN

Deaminase Activity on ssDNA. A USER (Uracil-Specific Excision Reagent) Enzyme-based assay for deamination was employed to test the activity of various deaminases on single-stranded DNA (ssDNA) substrates. USER Enzyme was obtained from New England Biolabs. An ssDNA substrate was provided with a target cytosine residue at different positions. Deamination of the ssDNA cytosine target residue results in conversion of the target cytosine to a uracil. The USER Enzyme excises the uracil base and cleaves the ssDNA backbone at that position, cutting the ssDNA substrate into two shorter fragments of DNA. In some assays, the ssDNA substrate is labeled on one end with a dye, e.g., with a 5′ Cy3 label (the * in the scheme below). Upon deamination, excision, and cleavage of the strand, the substrate can be subjected to electrophoresis, and the substrate and any fragment released from it can be visualized by detecting the label. Where Cy5 is images, only the fragment with the label will be visible via imaging.

In one USER Enzyme assay, ssDNA substrates were used that matched the target sequences of the various deaminases tested. Expression cassettes encoding the deaminases tested were inserted into a CMV backbone plasmid that has been used previously in the lab (Addgene plasmid 52970). The deaminase proteins were expressed using a TNT Quick Coupled Transcription/Translation System (Promega) according to the manufacturers recommendations. After 90 min of incubation, 5 mL of lysate was incubated with 5′ Cy3-labeled ssDNA substrate and 1 unit of USER Enzyme (NEB) for 3 hours. The DNA was resolved on a 10% TBE PAGE gel and the DNA was imaged using Cy-dye imaging. A schematic representation of the USER Enzyme assay is shown in FIG. 41.

FIG. 1 shows the deaminase activity of the tested deaminases on ssDNA substrates, such as Doench 1, Doench 2, G7′ and VEGF Target 2. The rAPOBEC1 enzyme exhibited a substantial amount of deamination on the single-stranded DNA substrate with a canonical NGG PAM, but not with a negative control non-canonical NNN PAM. Cas9 fusion proteins with APOBEC family deaminases were generated. The following fusion architectures were constructed and tested on ssDNA:

rAPOBEC1-GGS-dCas9 primary sequence (SEQ ID NO: 611) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW

rAPOBEC1-(GGS)₃-dCas9 primary sequence (SEQ ID NO: 612) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW

(SEQ ID NO: 613)

VDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFI EKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLEL YCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

(SEQ ID NO: 614)

ETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKH VEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYH HADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRL YVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

(SEQ ID NO: 615) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW

FIG. 2 shows that the N-terminal deaminase fusions showed significant activity on the single stranded DNA substrates. For this reason, only the N-terminal architecture was chosen for further experiments.

FIG. 3 illustrates double stranded DNA substrate binding by deaminase-dCas9:sgRNA complexes. A number of double stranded deaminase substrate sequences were generated. The sequences are provided below. The structures according to FIG. 3 are identified in these sequences (36 bp: underlined, sgRNA target sequence: bold; PAM: boxed; 21 bp: italicized). All substrates were labeled with a 5′-Cy3 label:

(SEQ ID NO: 616) 2:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGTC CCGCGGATTTATTTATTT

(SEQ ID NO: 617) 3:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCT TCCGCGGATTTATTTATT

(SEQ ID NO: 618) 4:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC TTCCGCGGATTTATTTAT

(SEQ ID NO: 619) 5:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC ATTCCGCGGATTTATTTA

(SEQ ID NO: 620) 6:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC TATTCCGCGGATTTATTT

(SEQ ID NO: 621) 7:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC TTATTCCGCGGATTTATT

(SEQ ID NO: 622) 8:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC ATTATTCCGCGGATTTAT

(SEQ ID NO: 623) 9:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC TATTATTCCGCGGATTTA

(SEQ ID NO: 624) 10:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC ATTATATTCCGCGGATTT

(SEQ ID NO: 625) 11:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC TATTATATTCCGCGGATT

(SEQ ID NO: 626) 12:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC TTATTATATTCCGCGGAT

(SEQ ID NO: 627) 13:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC ATTATTATATTCCGCGGA

(SEQ ID NO: 628) 14:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC TATTATTATATTCCGCGG

(SEQ ID NO: 629) 15:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC ATTATTATTATTACCGCG

(SEQ ID NO: 630) 18:GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC ATTATTATTATTATTACC

“-”: (SEQ ID NO: 631) GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGTA ATATTAATTTATTTATTTAA

(SEQ ID NO: 632) 8U:GTAGGTAGTTAGGATGAATGGAAGGTTGGTGTAG ATTATTATCUGCGGATTTA

*In all substrates except for “8U”, the top strand in FIG. 3 is the complement of the sequence specified here. In the case of “8U”, there is a “G” opposite the U.

FIG. 4 shows the results of a double stranded DNA Deamination Assay. The fusions were expressed and purified with an N-terminal His6 tag via both Ni-NTA and sepharose chromatography. In order to assess deamination on dsDNA substrates, the various dsDNA substrates shown on the previous slide were incubated at a 1:8 dsDNA:fusion protein ratio and incubated at 37° C. for 2 hours. Once the dCas9 portion of the fusion binds to the DNA it blocks access of the USER enzyme to the DNA. Therefore, the fusion proteins were denatured following the incubation and the dsDNA was purified on a spin column, followed by incubation for 45 min with the USER Enzyme and resolution of the resulting DNA substrate and substrate fragments on a 10% TBE-urea gel.

FIG. 5 demonstrates that Cas9 fusions can target positions 3-11 of double-stranded DNA target sequences (numbered according to the schematic in FIG. 3). Upper Gel: 1 μM rAPOBEC1-GGS-dCas9, 125 nM dsDNA, 1 eq sgRNA. Mid Gel: 1 μM rAPOBEC1-(GGS)₃-dCas9, 125 nM dsDNA, 1 eq sgRNA. Lower Gel: 1.85 μM rAPOBEC1-XTEN-dCas9, 125 nM dsDNA, 1 eq sgRNA. Based on the data from these gels, positions 3-11 (according to the numbering in FIG. 3) are sufficiently exposed to the activity of the deaminase to be targeted by the fusion proteins tested. Access of the deaminase to other positions is most likely blocked by the dCas9 protein.

The data further indicates that a linker of only 3 amino acids (GGS) is not optimal for allowing the deaminase to access the single stranded portion of the DNA. The 9 amino acid linker [(GGS)₃] (SEQ ID NO: 596) and the more structured 16 amino acid linker (XTEN) allow for more efficient deamination.

FIG. 6 demonstrates that the correct guide RNA, e.g., the correct sgRNA, is required for deaminase activity. The gel shows that fusing the deaminase to dCas9, the deaminase enzyme becomes sequence specific (e.g., using the fusion with an eGFP sgRNA results in no deamination), and also confers the capacity to the deaminase to deaminate dsDNA. The native substrate of the deaminase enzyme is ssDNA, and no deamination occurred when no sgRNA was added. This is consistent with reported knowledge that APOBEC deaminase by itself does not deaminate dsDNA. The data indicates that Cas9 opens the double-stranded DNA helix within a short window, exposing single-stranded DNA that is then accessible to the APOBEC deaminase for cytidine deamination. The sgRNA sequences used are provided below. sequences (36 bp: underlined, sgRNA target sequence: bold; PAM: boxed; 21 bp: italicized)

DNA Sequence 8:

(SEQ ID NO: 633) 5′-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC ATTATTCCGCGGATTTATT

Correct sgRNA sequence (partial 3′ sequence): (SEQ ID NO: 634) 5′-AUUAUUCCGCGGAUUUAUUUGUUUUAGAGCUAG...-3′ eGFP sgRNA sequence (partial 3′-sequence): (SEQ ID NO: 635) 5′-CGUAGGCCAGGGUGGUCACGGUUUUAGAGCUAG...-3′

Example 2: Deamination of DNA Target Sequence

Exemplary deamination targets. The dCas9:deaminase fusion proteins described herein can be delivered to a cell in vitro or ex vivo or to a subject in vivo and can be used to effect C to T or G to A transitions when the target nucleotide is in positions 3-11 with respect to a PAM. Exemplary deamination targets include, without limitation, the following: CCR5 truncations: any of the codons encoding Q93, Q102, Q186, R225, W86, or Q261 of CCR5 can be deaminated to generate a STOP codon, which results in a nonfunctional truncation of CCR5 with applications in HIV treatment. APOE4 mutations: mutant codons encoding C11R and C57R mutant APOE4 proteins can be deaminated to revert to the wild-type amino acid with applications in Alzheimer's treatment. eGFP truncations: any of the codons encoding Q158, Q184, Q185 can be deaminated to generate a STOP codon, or the codon encoding M1 can be deaminated to encode I, all of which result in loss of eGFP fluorescence, with applications in reporter systems. eGFP restoration: a mutant codon encoding T65A or Y66C mutant GFP, which does not exhibit substantial fluorescence, can be deaminated to restore the wild-type amino acid and confer fluorescence. PIK3CA mutation: a mutant codon encoding K111E mutant PIK3CA can be deaminated to restore the wild-type amino acid residue with applications in cancer. CTNNB1 mutation: a mutant codon encoding T41A mutant CTNNB1 can be deaminated to restore the wild-type amino acid residue with applications in cancer. HRAS mutation: a mutant codon encoding Q61R mutant HRAS can be deaminated to restore the wild-type amino acid residue with applications in cancer. P53 mutations: any of the mutant codons encoding Y163C, Y236C, or N239D mutant p53 can be deaminated to encode the wild type amino acid sequence with applications in cancer. The feasibility of deaminating these target sequences in double-stranded DNA is demonstrated in FIGS. 7 and 8. FIG. 7 illustrates the mechanism of target DNA binding of in vivo target sequences by deaminase-dCas9:sgRNA complexes.

FIG. 8 shows successful deamination of exemplary disease-associated target sequences. Upper Gel: CCR5 Q93: coding strand target in pos. 10 (potential off-targets at positions 2, 5, 6, 8, 9); CCR5 Q102: coding strand target in pos. 9 (potential off-targets at positions 1, 12, 14); CCR5 Q186: coding strand target in pos. 9 (potential off-targets at positions 1, 5, 15); CCR5 R225: coding strand target in pos. 6 (no potential off-targets); eGFP Q158: coding strand target in pos. 5 (potential off-targets at positions 1, 13, 16); eGFP Q184/185: coding strand target in pos. 4 and 7 (potential off-targets at positions 3, 12, 14, 15, 16, 17, 18); eGFP M1: template strand target in pos. 12 (potential off-targets at positions 2, 3, 7, 9, 11) (targets positions 7 and 9 to small degree); eGFP T65A: template strand target in pos. 7 (potential off-targets at positions 1, 8, 17); PIK3CA K111E: template strand target in pos. 2 (potential off-targets at positions 5, 8, 10, 16, 17); PIK3CA K111E: template strand target in pos. 13 (potential off-targets at positions 11, 16, 19) X. Lower Gel: CCR5 W86: template strand target in pos. 2 and 3 (potential off-targets at positions 1, 13) X; APOE4 C₁₁R: coding strand target in pos. 11 (potential off-targets at positions 7, 13, 16, 17); APOE4 C₅₇R: coding strand target in pos. 5) (potential off-targets at positions 7, 8, 12); eGFP Y66C: template strand target in pos. 11 (potential off-targets at positions 1, 4,6, 8, 9, 16); eGFP Y66C: template strand target in pos. 3 (potential off-targets at positions 1, 8, 17); CCR5 Q261: coding strand target in pos. 10 (potential off-targets at positions 3, 5, 6, 9, 18); CTNNB1 T41A: template strand target in pos. 7 (potential off-targets at positions 1, 13, 15, 16) X; HRAS Q61R: template strand target in pos. 6 (potential off-targets at positions 1, 2, 4, 5, 9, 10, 13); p53 Y163C: template strand target in pos. 6 (potential off-targets at positions 2, 13, 14); p53 Y236C: template strand target in pos. 8 (potential off-targets at positions 2, 4); p53 N239D: template strand target in pos. 4 (potential off-targets at positions 6, 8). Exemplary DNA sequences of disease targets are provided below (PAMs (5′-NGG-3′) and target positions are boxed):

(SEQ ID NO: 636) CCR5 Q93: 5′-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTAACTAT GCTGCCGCC

(SEQ ID NO: 637) CCR5 Q102: 5′-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAAATA CAATGTGT

(SEQ ID NO: 638) CCR5 Q186: 5'-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTATTTTC CATACAGT

(SEQ ID NO: 639) CCR5 R225: 5′-Cy3-

(SEQ ID NO: 640) CCR5 W86: 5′-Cy3-

(SEQ ID NO: 641) CCR5 Q261: 5′-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTATCCTG AACACCTT

(SEQ ID NO: 642) APOE4 C11R: 5′-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTAGACAT GGAGGAC

(SEQ ID NO: 643) APOE4 C57R: 5′-Cy3-

(SEQ ID NO: 644) eGFP Q158: 5′-Cy3-

(SEQ ID NO: 645) eGFP Q184/185: 5′-Cy3-

(SEQ ID NO: 646) eGFP M1: 5′-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTACCTCG CCCTTGCTCA

(SEQ ID NO: 647) eGFP T65A: 5′-Cy3-

(SEQ ID NO: 648) eGFP Y66C: 5'-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAAGCA CTGCACTC

(SEQ ID NO: 649) eGFP Y66C: 5′-Cy3-

(SEQ ID NO: 650) P1K3CA K111E: 5′-Cy3-

(SEQ ID NO: 651) P1K3CA K111E: 5′-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTATTCTC GATTG

(SEQ ID NO: 652) CTNNB1 T41A: 5′-Cy3- GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAGGAG CTGTGG

(SEQ ID NO: 653) HRAS Q61R: 5′-Cy3-

(SEQ ID NO: 654) p53 Y163C: 5′-Cy3-

(SEQ ID NO: 655) p53 Y236C: 5′-Cy3-

(SEQ ID NO: 656) p53 N239D: 5′-Cy3-

Example 3: Uracil Glycosylase Inhibitor Fusion Improves Deamination Efficiency

Direct programmable nucleobase editing efficiencies in mammalian cells by dCas9:deaminase fusion proteins can be improved significantly by fusing a uracil glycosylase inhibitor (UGI) to the dCas9:deaminase fusion protein.

FIG. 9 shows in vitro C→T editing efficiencies in human HEK293 cells using rAPOBEC1-XTEN-dCas9:

(SEQ ID NO: 657) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWV RLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK SGSETP

KV Protospacer sequences were as follows:

EMX1:

(SEQ ID NO: 293) FANCF:

(SEQ ID NO: 294) HEK293 site 2:

(SEQ ID NO: 295) HEK293 site 3:

(SEQ ID NO: 296) HEK293 site 4:

(SEQ ID NO: 297) RNF2:

(SEQ ID NO: 298) *PAMs are boxed, residues within target window (positions 3-11) are numbered and bolded.

FIG. 10 demonstrates that C→T editing efficiencies on the same protospacer sequences in HEK293T cells are greatly enhanced when a UGI domain is fused to the rAPOBEC1:dCas9 fusion protein.

(SEQ ID NO: 658) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWV RLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK SGSETP

IIEKETGKOLVICIESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAP EYKPWALVICIDSNGENKIKML SGGSPKKKRKV

The percentages in FIGS. 9 and 10 are shown from sequencing both strands of the target sequence. Because only one of the strands is a substrate for deamination, the maximum possible deamination value in this assay is 50%. Accordingly, the deamination efficiency is double the percentages shown in the tables. E.g., a value of 50% relates to deamination of 100% of double-stranded target sequences. When a uracil glycosylase inhibitor (UGI) was fused to the dCas9:deaminase fusion protein (e.g., rAPOBEC1-XTEN-dCas9-[UGI]-NLS), a significant increase in editing efficiency in cells was observed. This result indicates that in mammalian cells, the DNA repair machinery that cuts out the uracil base in a U:G base pair is a rate-limiting process in DNA editing. Tethering UGI to the dVas9:deaminase fusion proteins greatly increases editing yields.

Without UGI, typical editing efficiencies in human cells were in the ˜2-14% yield range (FIG. 9 and FIG. 10, “XTEN” entries). With UGI (FIG. 10, “UGI” entries) the editing was observed in the ˜6-40% range. Using a UGI fusion is thus more efficient than the current alternative method of correcting point mutations via HDR, which also creates an excess of indels in addition to correcting the point mutation. No indels resulting from treatment with the cas9:deaminase:UGI fusions were observed.

Example 4: Direct, Programmable Conversion of a Target Nucleotide in Genomic DNA without Double-Stranded DNA Cleavage

Current genome-editing technologies introduce double-stranded DNA breaks at a target locus of interest as the first step to gene correction.^(39,40) Although most genetic diseases arise from mutation of a single nucleobase to a different nucleobase, current approaches to revert such changes are very inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus as a consequence of the cellular response to double-stranded DNA breaks.^(39,40) Reported herein is the development of nucleobase editing, a new strategy for genome editing that enables the direct conversion of one target nucleobase into another in a programmable manner, without requiring double-stranded DNA backbone cleavage. Fusions of CRISPR/Cas9 were engineered and the cytidine deaminase enzyme APOBEC1 that retain the ability to be programmed with a guide RNA, do not induce double-stranded DNA breaks, and mediate the direct conversion of cytidine to uracil, thereby effecting a C→T (or G→A) substitution following DNA replication, DNA repair, or transcription if the template strand is targeted. The resulting “nucleobase editors” convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease in vitro. In four transformed human and murine cell lines, second- and third-generation nucleobase editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, respectively, can overcome the cellular DNA repair response to nucleobase editing, resulting in permanent correction of up to 37% or (˜15-75%) of total cellular DNA in human cells with minimal (typically ≤1%) indel formation. In contrast, canonical Cas9-mediated HDR on the same targets yielded an average of 0.7% correction with 4% indel formation. Nucleobase editors were used to revert two oncogenic p53 mutations into wild-type alleles in human breast cancer and lymphoma cells, and to convert an Alzheimer's Disease associated Arg codon in ApoE4 into a non-disease-associated Cys codon in mouse astrocytes. Base editing expands the scope and efficiency of genome editing of point mutations.

The clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive immune system that has been adapted to mediate genome engineering in a variety of organisms and cell lines.⁴¹ CRISPR/Cas9 protein-RNA complexes localize to a target DNA sequence through base pairing with a guide RNA, and natively create a DNA double-stranded break (DSB) at the locus specified by the guide RNA. In response to DSBs, endogenous DNA repair processes mostly result in random insertions or deletions (indels) at the site of DNA cleavage through non-homologous end joining (NHEJ). In the presence of a homologous DNA template, the DNA surrounding the cleavage site can be replaced through homology-directed repair (HDR). When simple disruption of a disease-associated gene is sufficient (for example, to treat some gain-of-function diseases), targeted DNA cleavage followed by indel formation can be effective. For most known genetic diseases, however, correction of a point mutation in the target locus, rather than stochastic disruption of the gene, is needed to address or study the underlying cause of the disease.⁶⁸

Motivated by this need, researchers have invested intense effort to increase the efficiency of HDR and suppress NHEJ. For example, a small-molecule inhibitor of ligase IV, an essential enzyme in the NHEJ pathway, has been shown to increase HDR efficiency.^(42,43) However, this strategy is challenging in post-mitotic cells, which typically down-regulate HDR, and its therapeutic relevance is limited by the potential risks of inhibiting ligase IV in non-target cells. Enhanced HDR efficiency can also be achieved by the timed delivery of Cas9-guide RNA complexes into chemically synchronized cells, as HDR efficiency is highly cell-cycle dependent.⁴⁴ Such an approach, however, is limited to research applications in cell culture since synchronizing cells is highly disruptive. Despite these developments, current strategies to replace point mutations using HDR in most contexts are very inefficient (typically ˜0.1 to 5%),^(42,43,45,46,75) especially in unmodified, non-dividing cells. In addition, HDR competes with NHEJ during the resolution of double-stranded breaks, and indels are generally more abundant outcomes than gene replacement. These observations highlight the need to develop alternative approaches to install specific modifications in genomic DNA that do not rely on creating double-stranded DNA breaks. A small-molecule inhibitor of ligase IV, an essential enzyme in the NHEJ pathway, has been shown to increase HDR efficiency.^(42,43) However, this strategy is challenging in post-mitotic cells, which typically down-regulate HDR, and its therapeutic relevance is limited by the potential risks of inhibiting ligase IV in non-target cells. Enhanced HDR efficiency can also be achieved by the timed delivery of Cas9-guide RNA complexes into chemically synchronized cells, as HDR efficiency is highly cell-cycle dependent.⁴⁴ Such an approach, however, is limited to research applications in cell culture since synchronizing cells is highly disruptive. In some cases, it is possible to design HDR templates such that the product of successful HDR contains mutations in the PAM sequence and therefore is no longer a substrate for subsequent Cas9 modification, increasing the overall yield of HDR products,⁷⁵ although such an approach imposes constraints on the product sequences. Recently, this strategy has been coupled to the use of ssDNA donors that are complementary to the non-target strand and high-efficiency ribonucleoprotein (RNP) delivery to substantially increase the efficiency of HDR, but even in these cases the ratio of HDR to NHEJ outcomes is relatively low (<2).⁸³

It was envisioned that direct catalysis of the conversion of one nucleobase to another at a programmable target locus without requiring DNA backbone cleavage could increase the efficiency of gene correction relative to HDR without introducing undesired random indels at the locus of interest. Catalytically dead Cas9 (dCas9), which contains Asp10Ala and His840Ala mutations that inactivate its nuclease activity, retains its ability to bind DNA in a guide RNA-programmed manner but does not cleave the DNA backbone.^(16,47) In principle, conjugation of dCas9 with an enzymatic or chemical catalyst that mediates the direct conversion of one nucleobase to another could enable RNA-programmed nucleobase editing. The deamination of cytosine (C) is catalyzed by cytidine deaminases²⁹ and results in uracil (U), which has the base pairing properties of thymine (T). dCas9 was fused to cytidine deaminase enzymes in order to test their ability to convert C to U at a guide RNA-specified DNA locus. Most known cytidine deaminases operate on RNA, and the few examples that are known to accept DNA require single-stranded DNA.⁴⁸ Recent studies on the dCas9-target DNA complex reveal that at least nine nucleotides of the displaced DNA strand are unpaired upon formation of the Cas9:guide RNA:DNA “R-loop” complex.¹² Indeed, in the structure of the Cas9 R-loop complex the first 11 nucleotides of the protospacer on the displaced DNA strand are disordered, suggesting that their movement is not highly restricted.⁷⁶ It has also been speculated that Cas9 nickase-induced mutations at cytosines in the non-template strand might arise from their accessibility by cellular cytidine deaminase enzymes.⁷⁷ Recent studies on the dCas9-target DNA complex have revealed that at least 26 bases on the non-template strand are unpaired when Cas9 binds to its target DNA sequence.⁴⁹ It was reasoned that a subset of this stretch of single-stranded DNA in the R-loop might serve as a substrate for a dCas9-tethered cytidine deaminase to effect direct, programmable conversion of C to U in DNA (FIG. 11A).

Four different cytidine deaminase enzymes (hAID, hAPOBEC3G, rAPOBEC1, and pmCDA1) were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and evaluated for ssDNA deamination. Of the four enzymes, rAPOBEC1 showed the highest deaminase activity under the tested conditions and was chosen for dCas9 fusion experiments (FIG. 36A). Although appending rAPOBEC1 to the C-terminus of dCas9 abolishes deaminase activity, fusion to the N-terminus of dCas9 preserves deaminase activity on ssDNA at a level comparable to that of the unfused enzyme. Four rAPOBEC1-dCas9 fusions were expressed and purified with linkers of different length and composition (FIG. 36B), and evaluated each fusion for single guide RNA (sgRNA)-programmed dsDNA deamination in vitro (FIGS. 11A to 11C and FIGS. 15A to 15D). Efficient, sequence-specific, sgRNA-dependent C to U conversion was observed in vitro (FIGS. 11A to 11C). Conversion efficiency was greatest using rAPOBEC1-dCas9 linkers over nine amino acids in length. The number of positions susceptible to deamination (the deamination “activity window”) increases with linker length was extended from three to 21 amino acids (FIGS. 36C to 36F15A to 15D). The 16-residue XTEN linker⁵⁰ was found to offer a promising balance between these two characteristics, with an efficient deamination window of approximately five nucleotides, from positions 4 to 8 within the protospacer, counting the end distal to the protospacer-adjacent motif (PAM) as position 1. The rAPOBEC1-XTEN-dCas9 protein served as the first-generation nucleobase editor (NBE1).

Elected were seven mutations relevant to human disease that in theory could be corrected by C to T nucleobase editing, synthesized double-stranded DNA 80-mers of the corresponding sequences, and assessed the ability of NBE1 to correct these mutations in vitro (FIGS. 16A to 16B). NBE1 yielded products consistent with efficient editing of the target C, or of at least one C within the activity window when multiple Cs were present, in six of these seven targets in vitro, with an average apparent editing efficiency of 44% (FIGS. 16A to 16B). In the three cases in which multiple Cs were present within the deamination window, evidence of deamination of some or all of these cytosines was observed. In only one of the seven cases tested were substantial yields of edited product observed (FIGS. 16A to 16B). Although the preferred sequence context for APOBEC1 substrates is reported to be CC or TC,⁵¹ it was anticipated that the increased effective molarity of the deaminase and its single-stranded DNA substrate mediated by dCas9 binding to the target locus may relax this restriction. To illuminate the sequence context generality of NBE1, its ability to edit a 60-mer double-stranded DNA oligonucleotide containing a single fixed C at position 7 within the protospacer was assayed, as well as all 36 singly mutated variants in which protospacer bases 1-6 and 8-13 were individually varied to each of the other three bases. Each of these 37 sequences were treated with 1.9 μM NBE1, 1.9 μM of the corresponding sgRNA, and 125 nM DNA for 2 h, similar to standard conditions for in vitro Cas9 assays⁵². High-throughput DNA sequencing (HTS) revealed 50 to 80% C to U conversion of targeted strands (25 to 40% of total sequence reads arising from both DNA strands, one of which is not a substrate for NBE1) (FIG. 12A). The nucleotides surrounding the target C had little effect on editing efficiency was independent of sequence context unless the base immediately 5′ of the target C is a G, in which case editing efficiency was substantially lower (FIGS. 12A to 12B). NBE1 activity in vitro was assessed on all four NC motifs at positions 1 through 8 within the protospacer (FIGS. 12A to 12B). In general NBE1 activity on substrates was observed to follow the order TC≥CC≥AC>GC, with maximum editing efficiency achieved when the target C is at or near position 7. In addition, it was observed that the nucleobase editor is highly processive, and will efficiently convert most of all Cs to Us on the same DNA strand within the 5-base activity window (FIG. 17).

While BE1 efficiently processes substrates in a test tube, in cells a tree of possible DNA repair outcomes determines the fate of the initial U:G product of base editing (FIG. 29A). To test the effectiveness of nucleobase editing in human cells, NBE1 codon usage was optimized for mammalian expression, appended a C-terminal nuclear localization sequence (NLS),⁵³ and assayed its ability to convert C to T in human cells on 14Cs in six well-studied target sites throughout the human genome (FIG. 37A).⁵⁴ The editable Cs were confirmed within each protospacer in vitro by incubating NBE1 with synthetic 80-mers that correspond to the six different genomic sites, followed by HTS (FIGS. 13A to 13C, FIG. 29B and FIG. 25). Next, HEK293T cells were transfected with plasmids encoding NBE1 and one of the six target sgRNAs, allowed three days for nucleobase editing to occur, extracted genomic DNA from the cells, and analyzed the loci by HTS. Although C to T editing in cells at the target locus was observed for all six cases, the efficiency of nucleobase editing was 1.1% to 6.3% or 0.8%-7.7% of total DNA sequences (corresponding to 2.2% to 12.6% of targeted strands), a 6.3-fold to 37-fold or 5-fold to 36-fold decrease in efficiency compared to that of in vitro nucleobase editing (FIGS. 13A to 13C, FIG. 29B and FIG. 25). It was observed that some base editing outside of the typical window of positions 4 to 8 when the substrate C is preceded by a T, which we attribute to the unusually high activity of APOBEC1 for TC substrates.⁴⁸

It was asked whether the cellular DNA repair response to the presence of U:G heteroduplex DNA was responsible for the large decrease in nucleobase editing efficiency in cells (FIG. 29A). Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells and initiates base excision repair (BER), with reversion of the U:G pair to a C:G pair as the most common outcome (FIG. 29A).⁵ Uracil DNA glycosylase inhibitor (UGI), an 83-residue protein from B. subtilis bacteriophage PBS1, potently blocks human UDG activity (IC₅₀=12 μM).⁵⁶ UGI was fused to the C-terminus of NBE1 to create the second-generation nucleobase editor NBE2 and repeated editing assays on all six genomic loci. Editing efficiencies in human cells were on average 3-fold higher with NBE2 than with NBE1, resulting in gene conversion efficiencies of up to 22.8% of total DNA sequenced (up to 45.6% of targeted strands) (FIGS. 13A to 13C and FIG. 29B). To test base editing in human cells, BE1 codon usage was optimized for mammalian expression and appended a C-terminal nuclear localization sequence (NLS).⁵³

Similar editing efficiencies were observed when a separate plasmid overexpressing UGI was co-transfected with NBE1 (FIGS. 18A to 18H). However, while the direct fusion of UGI to NBE1 resulted in no significant increase in C to T mutations at monitored non-targeted genomic locations, overexpression of unfused UGI detectably increased the frequency of C to T mutations elsewhere in the genome (FIGS. 18A to 18H). The generality of NBE2-mediated nucleobase editing was confirmed by assessing editing efficiencies on the same six genomic targets in U2OS cells, and observed similar results with those in HEK293T cells (FIG. 19). Importantly, NBE2 typically did not result in any detectable indels (FIG. 13C and FIG. 29C), consistent with the known mechanistic dependence of NHEJ on double-stranded DNA breaks.^(57,78) Together, these results indicate that conjugating UGI to NBE1 can greatly increase the efficiency of nucleobase editing in human cells.

The permanence of nucleobase editing in human cells was confirmed by monitoring editing efficiencies over multiple cell divisions in HEK293T cells at two of the tested genomic loci. Genomic DNA was harvested at two time points: three days after transfection with plasmids expressing NBE2 and appropriate sgRNAs, and after passaging the cells and growing them for four additional days (approximately five subsequent cell divisions). No significant change in editing efficiency was observed between the non-passaged cells (editing observed in 4.6% to 6.6% of targeted strands for three different target Cs) and passaged cells (editing observed in 4.6% to 6.4% of targeted strands for the same three target Cs), confirming that the nucleobase edits became permanent following cell division (FIG. 20). Indels will on rare occasion arise from the processing of U:G lesions by cellular repair processes, which involve single-strand break intermediates that are known to lead to indels.⁸⁴ Given that several hundred endogenous U:G lesions are generated every day per human cell from spontaneous cytidine deaminase,⁸⁵ it was anticipate that the total indel frequency from U:G lesion repair is unlikely to increase from BE1 or BE2 activity at a single target locus.

To further increase the efficiency of nucleobase editing in cells, it was anticipated that nicking the non-edited strand may result in a smaller fraction of edited Us being removed by the cell, since eukaryotic mismatch repair machinery uses strand discontinuity to direct DNA repair to any broken strand of a mismatched duplex (FIG. 29A).^(58,79,80) The catalytic His residue was restored at position 840 in the Cas9 HNH domain,^(47,59) resulting in the third-generation nucleobase editor NBE3 that nicks the non-edited strand containing a G opposite the targeted C, but does not cleave the target strand containing the C. Because NBE3 still contains the Asp10Ala mutation in Cas9, it does not induce double-stranded DNA cleavage. This strategy of nicking the non-edited strand augmented nucleobase editing efficiency in human cells by an additional 1.4- to 4.8-fold relative to NBE2, resulting in up to 36.3% of total DNA sequences containing the targeted C to T conversion on the same six human genomic targets in HEK293T cells (FIGS. 13A to 13C and FIG. 29B). Importantly, only a small frequency of indels, averaging 0.8% (ranging from 0.2% to 1.6% for the six different loci), was observed from NBE3 treatment (FIG. 13C, FIG. 29C, and FIG. 34). In contrast, when cells were treated with wild-type Cas9, sgRNA, and a single-stranded DNA donor template to mediate HDR at three of these loci C to T conversion efficiencies averaging only 0.7% were observed, with much higher relative indel formation averaging 3.9% (FIGS. 13A to 13C and FIG. 29C). The ratio of allele conversion to NHEJ outcomes averaged >1,000 for BE2, 23 for BE3, and 0.17 for wild-type Cas9 (FIG. 3c ). We confirmed the permanence of base editing in human cells by monitoring editing efficiencies over multiple cell divisions in HEK293T cells at the HEK293 site 3 and 4 genomic loci (FIG. 38). These results collectively establish that nucleobase editing can effect much more efficient targeted single-base editing in human cells than Cas9-mediated HDR, and with much less (NBE3) or no (NBE2) indel formation.

Next, the off-target activity of NBE1, NBE2, and NBE3 in human cells was evaluated. The off-target activities of Cas9, dCas9, and Cas9 nickase have been extensively studied (FIGS. 23 to 24 and 31 to 33).^(54,60-62) Because the sequence preference of rAPOBEC1 has been shown to be independent of DNA bases more than one base from the target C,⁶³ consistent with the sequence context independence observed in FIGS. 12A to 12B, it was assumed that potential off-target activity of nucleobase editors arises from off-target Cas9 binding. Since only a fraction of Cas9 off-target sites will have a C within the active window for nucleobase editing, off-target nucleobase editing sites should be a subset of the off-target sites of canonical Cas9 variants. For each of the six sites studied, the top ten known Cas9 off-target loci in human cells that were previously determined using the GUIDE-seq method were sequenced (FIGS. 23 to 27 and 31 to 33).^(54, 61) Detectable off-target nucleobase editing at only a subset (16/34, 47% for NBE1 and NBE2, and 17/34, 50% for NBE3) of known dCas9 off-target loci was observed. In all cases, the off-target base-editing substrates contained a C within the five-base target window. In general, off-target C to T conversion paralleled off-target Cas9 nuclease-mediated genome modification frequencies (FIGS. 23 to 27). Also monitored were C to T conversions at 2,500 distinct cytosines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8×10⁶ cells, and observed no detectable increase in C to T conversions at any of these other sites upon NBE1, NBE2, or NBE3 treatment compared to that of untreated cells (FIG. 28). Taken together, these findings suggest that off-target substrates of nucleobase editors include a subset of Cas9 off-target substrates, and that nucleobase editors in human cells do not induce untargeted C to T conversion throughout the genome at levels that can be detected by the methods used here. No substantial change was observed in editing efficiency between non-passaged HEK293T cells (editing observed in 1.8% to 2.6% of sequenced strands for the three target Cs with BE2, and 6.2% to 14.3% with BE3) and cells that had undergone approximately five cell divisions after base editing (editing observed in 1.9% to 2.3% of sequenced strands for the same target Cs with BE2, and 6.4% to 14.5% with BE3), confirming that base edits in these cells are durable (Extended Data FIG. 6).

Finally, the potential of nucleobase editing to correct three disease-relevant mutations in mammalian cells was tested. The apolipoprotein E gene variant APOE4 encodes two Arg residues at amino acid positions 112 and 158, and is the largest and most common genetic risk factor for late-onset Alzheimer's disease.⁶⁴ ApoE variants with Cys residues in positions 112 or 158, including APOE2 (Cys112/Cys158), APOE3 (Cys112/Arg158), and APOE3′ (Arg112/Cys158) have been shown⁶⁵ or are presumed⁸¹ to confer substantially lower Alzheimer's disease risk than APOE4. Encouraged by the ability of NBE1 to convert APOE4 to APOE3′ in vitro (FIGS. 16A to 16B), this conversion was attempted in immortalized mouse astrocytes in which the endogenous murine APOE gene has been replaced by human APOE4 (Taconic). DNA encoding NBE3 and an appropriate sgRNA was delivered into these astrocytes by nucleofection (nucleofection efficiency of 25%), extracted genomic DNA from all treated cells two days later, and measured editing efficiency by HTS. Conversion of Arg158 to Cys158 was observed in 58-75% of total DNA sequencing reads (44% of nucleofected astrocytes) (FIGS. 14A to 14C and FIG. 30A). Also observed was 36-50% editing of total DNA at the third position of codon 158 and 38-55% editing of total DNA at the first position of Leu159, as expected since all three of these Cs are within the active nucleobase editing window. However, neither of the other two C→T conversions results in a change in the amino acid sequence of the ApoE3′ protein since both TGC and TGT encode Cys, and both CTG and TTG encode Leu. From >1,500,000 sequencing reads derived from 1×10⁶ cells evidence of 1.7% indels at the targeted locus following NBE3 treatment was observed (FIG. 35). In contrast, identical treatment of astrocytes with wt Cas9 and donor ssDNA resulted in 0.1-0.3% APOE4 correction and 26-40% indels at the targeted locus, efficiencies consistent with previous reports of single-base correction using Cas9 and HDR^(45,75) (FIG. 30A and FIG. 40A). Astrocytes treated identically but with an sgRNA targeting the VEGFA locus displayed no evidence of APOE4 base editing (FIG. 34 and FIG. 40A). These results demonstrate how nucleobase editors can effect precise, single-amino acid changes in the coding sequence of a protein as the major product of editing, even when their processivity results in more than one nucleotide change in genomic DNA. The off-target activities of Cas9, dCas9, and Cas9 nickase have been extensively studied.^(54, 60-62) In general, off-target C to T conversions by BE1, BE2, and BE3 paralleled off-target Cas9 nuclease-mediated genome modification frequencies.

The dominant-negative p53 mutations Tyr163Cys and Asn239Asp are strongly associated with several types of cancer.⁶⁶⁻⁶⁷ Both of these mutations can be corrected by a C to T conversion on the template strand (FIGS. 16A to 16B). A human breast cancer cell line homozygous for the p53 Tyr163Cys mutation (HCC1954 cells) was nucleofected with DNA encoding NBE3 and an sgRNA programmed to correct Tyr163Cys. Because the nucleofection efficiency of HCC1954 cells was <10%, a plasmid expressing IRFP was co-nucleofected into these cells to enable isolation of nucleofected cells by fluorescence-activated cell sorting two days after treatment. HTS of genomic DNA revealed correction of the Tyr163Cys mutation in 7.6% of nucleofected HCC1954 cells (FIG. 30B and FIG. 40A to 40B). Also nucleofected was a human lymphoma cell line that is heterozygous for p53 Asn239Asp (ST486 cells) with DNA encoding NBE2 and an sgRNA programmed to correct Asn239Asp with 92% nucleofection efficiency). Correction of the Asn239Asp mutation was observed in 11% of treated ST486 cells (12% of nucleofected ST486 cells). Consistent with the findings in HEK cells, no indels were observed from the treatment of ST486 cells with NBE2, and 0.6% indel formation from the treatment of HCC1954 cells with NBE3. No other DNA changes within at least 50 base pairs of both sides of the protospacer were detected at frequencies above that of untreated controls out of >2,000,000 sequencing reads derived from 2×10⁵ cells (FIGS. 14A to 14C, FIG. 30B and Table 1). These results collectively represent the conversion of three disease-associated alleles in genomic DNA into their wild-type forms with an efficiency and lack of other genome modification events that is, to our knowledge, not currently achievable using other methods.

To illuminate the potential relevance of nucleobase editors to address human genetic diseases, the NCBI ClinVar database⁶⁸ was searched for known genetic diseases that could in principle be corrected by this approach. ClinVar was filtered by first examining only single nucleotide polymorphisms (SNPs), then removing any nonpathogenic variants. Out of the 24,670 pathogenic SNPs, 3,956 are caused by either a T to C, or an A to G, substitution. This list was further filtered to only include variants with a nearby NGG PAM that would position the SNP within the deamination activity window, resulting in 1,089 clinically relevant pathogenic gene variants that could in principle be corrected by the nucleobase editors described here (FIG. 21 and Table 1). To illuminate the potential relevance of base editors to address human genetic diseases, the NCBI ClinVar database⁶⁸ was searched for known genetic diseases that could in principle be corrected by this approach. ClinVar was filtered by first examining only single nucleotide polymorphisms (SNPs), then removing any non-pathogenic variants. Out of the 24,670 pathogenic SNPs, 3,956 are caused by either a T to C, or an A to G, substitution. This list was further filtered to only include variants with a nearby NGG PAM that would position the SNP within the deamination activity window, resulting in 911 clinically relevant pathogenic gene variants that could in principle be corrected by the base editors described here. Of these, 284 contain only one C within the base editing activity window. A detailed list of these pathogenic mutations can be found in Table 1.

TABLE 1 List of 911 base-editable gene variants associated with human disease with an NGG PAM (SEQ ID NOs: 747 to 1868 appear from top to bottom below, respectively). The “Y” in the protospacer and PAM sequences indicates the base to be edited, e.g., C. (SEQ ID NOs: 747 to 1868 appear from top to bottom below, respectively) Protospacer dbSNP # Genotype and PAM sequence(s) Associated genetic disease 755445790 NM_000391.3(TPP1):c.887-10A>G TTTYTTTTTTTTTTTTTTTGAGG Ceroid lipofuscinosis, neuronal, 2 113994167 NM_000018.3(ACADVL):c.848T>C TTTGYGGTGGAGAGGGGCTTCGG, Very long chain acyl-CoA (p.Val283Ala) TTGYGGTGGAGAGGGGCTTCGGG dehydrogenase deficiency 119470018 NM_024996.5(GFM1):c.521A>G TTGYTAATAAAAGTTAGAAACGG Combined oxidative phosphorylation (p.Asn174Ser) deficiency 1 115650537 NM_000426.3(LAMA2):c.8282T>C TTGAYAGGGAGCAAGCAGTTCGG, Merosin deficient congenital (p.Ile2761Thr) TGAYAGGGAGCAAGCAGTTCGGG muscular dystrophy 587777752 NM_014946.3(SPAST):c.1688- TTCYGTAAAACATAAAAGTCAGG Spastic paraplegia 4, autosomal dominant 794726821 NM_001165963.1(SCN1A):c.4055T>C TTCYGGTTTGTCTTATATTCTGG Severe myoclonic epilepsy in infancy (p.Leu1352Pro) 397514745 NM_001130089.1(KARS):c.517T>C CTTCYATGATCTTCGAGGAGAGG, Deafness, autosomal recessive 89 (p.Tyr173His) TTCYATGATCTTCGAGGAGAGG G 376960358 NM_001202.3(BMP4):c.362A>G TTCGTGGYGGAAGCTCCTCACGG Microphthalmia syndromic 6 (p.His121Arg) 606231280 NM_001287223.1(SCN11A):c.1142T>C CTTCAYTGTGGTCATTTTCCTGG, Episodic pain syndrome, familial, 3 (p.Ile381Thr) TTCAYTGTGGTCATTTTCCTGG G 387906735 m.608A>G TTCAGYGTATTGCTTTGAGGAGG 199474663 m.3260A>G TTAAGTTYTATGCGATTACCGGG Cardiomyopathy with or without skeletal myopathy 104894962 NM_003413.3(ZIC3):c.1213A>G TGTGTTYGCGCAGGGAGCTCGGG, Heterotaxy, visceral, X-linked (p.Lys405Glu) ATGTGTTYGCGCAGGGAGCTCG G 796053181 NM_021007.2(SCN2A):c.1271T>C TGTGGYGGCCATGGCCTATGAGG not provided (p.Val424Ala) 267606788 NM_000129.3(F13A1):c.728T>C TGTGAYGGACAGAGCACAAATGG Factor xiii, a subunit, deficiency of (p.Met243Thr) 397514503 NM_003863.3(DPM2):c.68A>G TGTAGYAGGTGAAGATGATCAGG Congenital disorder of glycosylation type (p.Tyr23Cys) 1u 104893973 NM_000416.2(IFNGR1):c.260T>C TGTAATAYTTCTGATCATGTTGG Disseminated atypical mycobacterial (p.Ile87Thr) infection, Mycobacterium tuberculosis, susceptibility to 121908466 NM_005682.6(ADGRG1):c.263A>G TGGYAGAGGCCCCTGGGGTCAGG Polymicrogyria, bilateral frontoparietal (p.Tyr88Cys) 147952488 NM_002437.4(MPV17):c.186+2T>C TGGYAAGTTCTCCCCTCAACAGG Navajo neurohepatopathy 21909537 NM_001145.4(ANG):c.121A>G TGGTTYGGCATCATAGTGCTGGG, Amyotrophic lateral sclerosis type 9 (p.Lys41Glu) GTGGTTYGGCATCATAGTGCTG G 121918489 NM_000141.4(FGFR2):c.1018T>C TGGGGAAYATACGTGCTTGGCGG, Crouzon syndrome (p.Tyr340His) GGGGAAYATACGTGCTTGGCGGG 121434463 m.12320A>G GAGTYGCACCAAAATTTTTGGGG, Mitochondrial myopathy GGAGTYGCACCAAAATTTTTGGG, TGGAGTYGCACCAAAATTTTTG G 121908046 NM_000403.3(GALE):c.101A>G TGGAAGYTATCGATGACCACAGG UDPglucose-4-epimerase deficiency (p.Asn34Ser) 431905512 NM_003764.3(STX11):c.173T>C TGCYGGTGGCCGACGTGAAGCGG Hemophagocytic lymphohistiocytosis, (p.Leu58Pro) familial, 4 121917905 NM_000124.3(ERCC6):c.2960T>C TGCYAAAAGACCCAAAACAAAGG Cerebro-oculo-facio-skeletal syndrome (p.Leu987Pro) 121918500 NM_000141.4(FGFR2):c.874A>G TGCTYGATCCACTGGATGTGGGG, Crouzon syndrome (p.Lys292Glu) GTGCTYGATCCACTGGATGTGGG, CGTGCTYGATCCACTGGATGTG G 60431989 NM_000053.3(ATP7B):c.3443T>C TGCTGAYTGGAAACCGTGAGTGG Wilson disease (p.Ile1148Thr) 78950939 NM_000250.1(MPO):c.518A>G GTGCGGYATTTGTCCTGCTCCGG, Myeloperoxidase deficiency (p.Tyr173Cys) TGCGGYATTTGTCCTGCTCCGG G 115677373 NM_201631.3(TGM5):c.763T>C TGCGGAGYGGACGGGCAGCGTGG Peeling skin syndrome, acral type (p.Trp255Arg) 5030804 NM_000551.3(VHL):c.233A>G GCGAYTGCAGAAGATGACCTGGG, Von Hippel-Lindau syndrome (p.Asn78Ser) TGCGAYTGCAGAAGATGACCTG G 397508328 NM_000492.3(CFTR):c.1A>G GCAYGGTCTCTCGGGCGCTGGGG, Cystic fibrosis (p.Met1Val) TGCAYGGTCTCTCGGGCGCTGGG, CTGCAYGGTCTCTCGGGCGCTGG 137853299 NM_000362.4(TIMP3):c.572A>G TGCAGYAGCCGCCCTTCTGCCGG Sorsby fundus dystrophy (p.Tyr191Cys) 121908549 NM_000334.4(SCN4A):c.3478A>G TGAYGGAGGGGATGGCGCCTAGG (p.Ile1160Val) 121909337 NM_001451.2(FOXF1):c.1138T>C TGATGYGAGGCTGCCGCCGCAGG Alveolar capillary dysplasia with (p.Ter380Arg) misalignment of pulmonary veins 281875320 NM_005359.5(SMAD4):c.1500A>G TGAGYATGCATAAGCGACGAAGG Myhre syndrome (p.Ile500Met) 730880132 NM_170707.3(LMNA):c.710T>C TGAGTYTGAGAGCCGGCTGGCGG Primary dilated cardiomyopathy (p.Phe237Ser) 281875322 NM_005359.5(SMAD4):c.1498A>G TGAGTAYGCATAAGCGACGAAGG Hereditary cancer-predisposing syndrome, (p.Ile500Val) Myhre syndrome 72556283 NM_000531.5(OTC):c.527A>G TGAGGYAATCAGCCAGGATCTGG not provided (p.Tyr176Cys) 74315311 NM_020435.3(WC2):c.857T>C TGAGAYGGCCCACCTGGGCTTGG, Leukodystrophy, hypomyelinating, 2 (p.Met286Thr) GAGAYGGCCCACCTGGGCTTGGG 121912495 NM_170707.3(LMNA):c.1139T>C TCTYGGAGGGCGAGGAGGAGAGG Congenital muscular dystrophy, LMNA-related (p.Leu380Ser) 128620184 NM_000061.2(BTK):c.1288A>G TCTYGATGGCCACGTCGTACTGG X-linked agammaglobulinemia (p.Lys430Glu) 118192252 NM_004519.3(KCNQ3):c.1403A>G TCTTTAYTGTTTAAGCCAACAGG Benign familial neonatal seizures 2, not (p.Asn468Ser) specified 121909142 NM_001300.5(KLF6):c.190T>C TCTGYGGACCAAAATCATTCTGG (p.Trp64Arg) 104895503 NM_001127255.1(NLRP7):c.2738A>G TCTGGYTGATACTCAAGTCCAGG Hydatidiform mole (p.Asn913Ser) 587783035 NM_000038.5(APC):c 1744-2A>G TCCYAGTAAGAAACAGAATATGG Familial adenomatous polyposis 72556289 NM_000531.5(OTC):c.541-2A>G TCCYAAAAGGCACGGGATGAAGG not provided 28937313 NM_005502.3(ABCA1):c.2804A>G TCCAYTGTGGCCCAGGAAGGAGG, Tangier disease (p.Asn935Ser) CGCTCCAYTGTGGCCCAGGAAGG 143246552 NM_001003811.1(TEX11):c.511A>G TCCAYGGTCAAGTCAGCCTCAGG, Spermatogenic failure, X-linked, 2 (p.Met171Val) CCAYGGTCAAGTCAGCCTCAGGG 587776451 NM_002049.3(GATA1):c.2T>C CTCCAYGGAGTTCCCTGGCCTGG, GATA-1-related thrombocytopenia (p.Met1Thr) TCCAYGGAGTTCCCTGGCCTGGG, with dyserythropoiesis CCAYGGAGTTCCCTGGCCTGGGG 121908403 NM_021102.3(SPINT2):c.488A>G TCCAYAGATGAAGTTATTGCAGG Diarrhea 3, secretory sodium, congenital, (p.Tyr163Cys) syndromic 281874738 NM_000495.4(COL4A5):c.438+2T>C CTCCAGYAAGTTATAAAATTTGG, Alport syndrome, X-linked recessive TCCAGYAAGTTATAAAATTTGG G 730880279 NM_030653.3(DDX11):c.2271+2T>C TCCAGGYGCGGGCGTCATGCTGG, Warsaw breakage syndrome CCAGGYGCGGGCGTCATGCTGGG 28940272 NM_017890.4(VPS13B):c.8978A>G TCAYTGATAAGCAGGGCCCAGGG, Cohen syndrome, not specified (p.Asn2993Ser) TTCAYTGATAAGCAGGGCCCAGG 137852375 NM_000132.3(F8):c.5372T>C TCAYGGTGAGTTAAGGACAGTGG Hereditary factor VIII deficiency disease (p.Met1791Thr) 11567847 NM_021961.5(TEAD1):c.1261T>C TCATATTYACAGGCTTGTAAAGG (p.Tyr?His) 786203989 NM_016069.9(PAM16):c.226A>G CATAGTYCTGCAGAGGAGAGGGG, Chondrodysplasia, megarbane-dagher-melki (p.Asn76Asp) TCATAGTYCTGCAGAGGAGAGGG type 587776437 NC_012920.1:m.9478T>C TCAGAAGYTTTTTTCTTCGCAGG Leigh disease 121912474 NM_000424.3(KRT5):c.20T>C TCAAGTGYGTCCTTCCGGAGCGG, Epidermolysis bullosa simplex, Koebner type (p.Val7Ala) CAAGTGYGTCCTTCCGGAGCGGG, AAGTGYGTCCTTCCGGAGCGGGG, AGTGYGTCCTTCCGGAGCGGGGG 104886461 NM_020533.2(MCOLN1):c.406-2A>G TACYGTGGGCAGAGAAGGGGAGG, Ganglioside sialidase deficiency AGGTACYGTGGGCAGAGAAGGGG, CAGGTACYGTGGGCAGAGAAGGG 104894275 NM_000317.2(PTS):c.155A>G TAAYTGTGCCCATGGCCATTTGG 6-pyruvoyl-tetrahydropterin synthase deficiency (p.Asn52Ser) 587777562 NM_015599.2(PGM3):c.737A>G TAAATGAYTGAGTTTGCCCTTGG Immunodeficiency 23 (p.Asn246Ser) 121964906 NM_000027.3(AGA):c.916T>C GTTATAYGTGCCAATGTGACTGG Aspartylglycosaminuria (p.Cys306Arg) 28941769 NM_000356.3(TCOF1):c.149A>G GTGTGTAYAGATGTCCAGAAGGG Treacher collins syndrome 1 (p.Tyr50Cys) 121434464 m.12297T>C GTCYTAGGCCCCAAAAATTTTGG Cardiomyopathy, mitochondrial 121908407 NM_054027.4(ANKH):c.143T>C GTCGAGAYGCTGGCCAGCTACGG, Chondrocalcinosis 2 (p.Met48Thr) TCGAGAYGCTGGCCAGCTACGGG 59151893 NM_000422.2(KRT17):c.275A>G GTCAYTGAGGTTCTGCATGGTGG, Pachyonychia congenita type 2 (p.Asn92Ser) GCGGTCAYTGAGGTTCTGCATGG 121909499 NM_002427.3(MMP13):c.272T>C GTCAYGAAAAAGCCAAGATGCGG, (p.Met91Thr) TCAYGAAAAAGCCAAGATGCGG G 61748478 NM_000552.3(VWF):c.2384A>G GTCAYAGTTCTGGCACGTTTTGG von Willebrand disease type 2N (p.Tyr795Cys) 387906889 NM_006796.2(AFG3L2):c.1847A>G GTAYAGAGGTATTGTTCTTTTGG Spastic ataxia 5, autosomal recessive (p.Tyr616Cys) 118203907 NM_000130.4(F5):c.5189A>G GTAGYAGGCCCAAGCCCGACAGG Factor V deficiency (p.Tyr1730Cys) 118203945 NM_013319.2(UBIAD1):c.305A>G GTAAGTGYTGACCAAATTACCGG Schnyder crystalline corneal dystrophy (p.Asn102Ser) 267607080 NM_005633.3(SOSI):c.1294T>C GGTYGGGAGGGAAAAGACATTGG Noonan syndrome 4, Rasopathy (p.Trp432Arg) 137852953 NM_012464.4(TLL1):c.1885A>G GGTTAYGGTGCCGTTAAGTTTGG Atrial septal defect 6 (p.Ile629Val) 118203949 NM_013319.2(UBIAD1):c.695A>G GGTGTTGYTGGAATGGAGAATGG Schnyder crystalline corneal dystrophy (p.Asn232Ser) 137852952 NM_012464.4(TLL1):c.713T>C GGGATTGYTGTTCATGAATTGGG Atrial septal defect 6 (p.Val238Ala) 41460449 m.3394T>C GGCYATATACAACTACGCAAAGG Leber optic atrophy 80357281 NM_007294.3(BRCA1):c.5291T>C GGGCYAGAAATCTGTTGCTATGG, Familial cancer of breast, Breast-ovarian (p.Leu1764Pro) GGCYAGAAATCTGTTGCTATGGG cancer, familial 1 5030764 NM_000174.4(GP9):c.182A>G GGCTGYTGTTGGCCAGCAGAAGG Bernard-Soulier syndrome type C (p.Asn61Ser) 72556282 NM_000531.5(OTC):c.526T>C GGCTGATYACCTCACGCTCCAGG, not provided (p.Tyr176His) GATYACCTCACGCTCCAGGTTGG 121913594 NM_000530.6(MPZ):c.242A>G GGCATAGYGGAAGATCTATGAGG Charcot-Marie-Tooth disease type 1B (p.His8lArg) 587777736 NM_017617.3(NOTCH1):c.1285T>C GGCAAGYGCATCAACACGCTGGG, Adams-Oliver syndrome 1, Adams- (p.Cys429Arg) GGGCAAGYGCATCAACACGCTGG Oliver syndrome 5 63750912 NM_016835.4(MAPT):c.1839T>C GGATAAYATCAAACACGTCCCGG, Frontotemporal dementia (p.Asn613=) GATAAYATCAAACACGTCCCGG G 121918075 NM_000371.3(TTR):c.401A>G GGAGYAGGGGCTCAGCAGGGCGG, Amyloidogenic transthyretin amyloidosis (p.Tyr134Cys) ATAGGAGYAGGGGCTCAGCAGGG 730882063 NM_004523.3(KIF11):c.2547+2T>C GGAGGYAATAACTTTGTAAGTGG Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation 397516156 NM_000257.3(MYH7):c.2546T>C GGAGAYGGCCTCCATGAAGGAGG Primary familial hypertrophic (p.Met849Thr) cardiomyopathy, 118204430 NM_000035.3(ALDOB):c.442T>C GGAAGYGGCGTGCTGTGCTGAGG Hereditary fructosuria (p.Trp148Arg) 200198778 NM_013382.5(POMT2):c.1997A>G GGAAGYAGTGGTGGAAGTAGAGG Congenital muscular dystrophy, Congenital (p.Tyr666Cys) muscular dystrophy-dystroglycanopathy with brain and eye anomalies, type A2, Muscular dystrophy, Congenital muscular dystrophy-dystroglycanopathy with mental retardation, type B2 754896795 NM_004006.2(DMD):c.6982A>T GCTTTTYTTCAAGCTGCCCAAGG Duchenne muscular dystrophy, Becker (p.Lys2328Ter) muscular dystrophy, Dilated cardiomyopathy 3B 148924904 NM_000546.5(TP53):c.488A>G GCTTGYAGATGGCCATGGCGCGG Hereditary cancer-predisposing syndrome (p.Tyr163Cys) 786204770 NM_016035.4(C0Q4):c.155T>C GCTGTYGGCCGCCGGCTCCGCGG COENZYME Q10 DEFICIENCY, PRIMARY, 7 (p.Leu52Ser) 121909520 NM_001100.3(ACTA1):c.350A>G CGGYTGGCCTTGGGATTGAGGGG, Nemaline myopathy 3 (p.Asn117Ser) GCGGYTGGCCTTGGGATTGAGGG, CGCGGYTGGCCTTGGGATTGAGG 587776879 NM_004656.3(BAP1):c.438-2A>G GCCYGGGGAAAAACAGAGTCAGG Tumor predisposition syndrome 727504434 NM_000501.3(ELN):c.890-2A>G GCCYGAAAACACAGCCACAGAGG Supravalvar aortic stenosis 119455953 NM_000391.3(TPP1):c.1093T>C GCCGGGYGTTGGTCTGTCTCTGG Ceroid lipofuscinosis, neuronal, 2 (p.Cys365Arg) 121964983 NM_000481.3(AMT):c.125A>G GCCAGGYGGAAGTCATAGAGCGG Non-ketotichyperglycinemia (p.His42Arg) 121908300 NM_001005741.2(GBA):c.751T>C GCCAGAYACTTTGTGAAGTAAGG, Gaucher disease, type 1 (p.Tyr251His) CCAGAYACTTTGTGAAGTAAGG 786205083 NM_003494.3(DYSF):c.3443-33A>G GCCAGAGYGAGTGGCTGGAGTGG Limb-girdle muscular dystrophy, type 2B 121908133 NM_175073.2(APTX):c.602A>G GCCAAYGGTAACGGGCCTTTGGG, Adult onset ataxia with oculomotor apraxia (p.His201Arg) AGCCAAYGGTAACGGGCCTTTGG 587777195 NM_005017.3(PCYT1A):c.571T>C GCATGYTTGCTCCAACACAGAGG Spondylometaphyseal dysplasia with cone-rod (p.Phe191Leu) dystrophy 431905520 NM_014714.3(IFT140):c.4078T>C CAAGCAGYGTGAGCTGCTCCTGG, Renal dysplasia, retinal pigmentary dystrophy, (p.Cys1360Arg) GCAGYGTGAGCTGCTCCTGGAGG cerebellar ataxia and skeletal dysplasia 121912889 NM_001844.4(COL2A1):c.4172A>G GCAGTGGYAGGTGATGTTCTGGG Spondyloperipheral dysplasia, Platyspondylic (p.Tyr1391Cys) lethal skeletal dysplasia Torrance type 137854492 NM_001363.4(DKC1):c.1069A>G GCAGGYAGAGATGACCGCTGTGG Dyskeratosis congenita X-linked (p.Thr357Ala) 121434362 NM_152783.4(D2HGDH):c.1315A>G GCAGGTYACCATCTCCTGGAGGG, D-2-hydroxyglutaric aciduria 1 (p.Asn439Asp) TGCAGGTYACCATCTCCTGGAGG 80338732 NM_002764.3(PRPS1):c.344T>C GCAAATAYGCTATCTGTAGCAGG Charcot-Marie-Tooth disease, X- (p.Met115Thr) linked recessive, type 5 387906675 NM_000313.3(PROS1):c.701A>G GATTAYATCTGTAGCCTTCGGGG, Thrombophilia due to protein S deficiency, (p.Tyr234Cys) AGATTAYATCTGTAGCCTTCGGG, autosomal recessive GAGATTAYATCTGTAGCCTTCGG 28935478 NM_000061.2(BTK):c.1082A>G GATGGYAGTTAATGAGCTCAGGG, (p.Tyr36ICys) TGATGGYAGTTAATGAGCTCAGG 201777056 NM_005050.3(ABCD4):c.956A>G GATGAGGYAGATGCACACAAAGG METHYLMALONIC ACIDURIA (p.Tyr319Cys) AND HOMOCYSTINURIA, cblJ 121918528 NM_000098.2(CPT2):c.359A>G GATAGGYACATATCAAACCAGGG, Carnitine palmitoyltransferase II (p.TyrI20Cys) AGATAGGYACATATCAAACCAG deficiency, infantile G 267607014 NM_002942.4(ROBO2):c.2834T>C GAGAYTGGAAATTTTGGCCGTGG Vesicoureteral reflux 2 (p.Ile945Thr) 281865192 NM_025114.3(CEP290):c.2991+1655 GATAYTCACAATTACAACTGGGG, Leber congenital amaurosis 10 A>G AGATAYTCACAATTACAACTGGG, GAGATAYTCACAATTACAACTG 386833492 NM_000112.3(SLC26A2):c.- GAGAGGYGAGAAGAGGGAAGCGG Diastrophic dysplasia 26+2T>C 587779773 NM_001101.3(ACTB):c.356T>C GAGAAGAYGACCCAGGTGAGTGG Baraitser-Winter syndrome 1 (p.Met119Thr) 121913512 NM_000222.2(KIT):c.1924A>G GACTTYGAGTTCAGACATGAGGG, (p.Lys642Glu) GGACTTYGAGTTCAGACATGAGG 28939072 NM_006329.3(FBLN5):c.506T>C GACAYTGATGAATGTCGCTATGG Age-related macular degeneration 3 (p.Ile169Thr) 104894248 NM_000525.3(KCNJ11):c.776A>G GACAYGGTAGATGATCAGCGGGG, Islet cell hyperplasia (p.His259Arg) TGACAYGGTAGATGATCAGCGGG, ATGACAYGGTAGATGATCAGCGG 387907132 NM_016464.4(TMEM138):c.287A>G GACAYGAAGGGAGATGCTGAGGG, Joubert syndrome 16 (p.His96Arg) AGACAYGAAGGGAGATGCTGAGG 121918170 NM_000275.2(OCA2):c.1465A>G GACATYTGGAGGGTCCCCGATGG Tyrosinase-positive oculocutaneous albinism (p.Asn489Asp) 122467173 NM_014009.3(FOXP3):c.970T>C GACAGAGYTCCTCCACAACATGG Insulin-dependent diabetes mellitus secretory (p.Phe324Leu) diarrhea syndrome 137852268 NM_000133.3(F9):c.1328T>C GAAYATATACCAAGGTATCCCGG Hereditary factor LX deficiency disease (p.Ile443Thr) 149054177 NM_001999.3(FBN2):c.3740T>C GAATGTAYGATAATGAACGGAGG not specified, Macular degeneration, early- (p.Met1247Thr) onset 137854488 NM_212482.1(FN1):c.2918A>G GAAGTAAYAGGTGACCCCAGGGG Glomerulopathy with fibronectin deposits 2 (p.Tyr973Cys) 786204027 NM_005957.4(MTHFR):c.1530+2T>C GAAGGYGTGGTAGGGAGGCACGG, Homocysteinemia due to MTHFR deficiency AAGGYGTGGTAGGGAGGCACGGG, AGGYGTGGTAGGGAGGCACGGGG 104894223 NM_012193.3(FZD4):c.766A>G GAAATAYGATGGGGCGCTCAGGG, Retinopathy of prematurity (p.Ile256Val) AGAAATAYGATGGGGCGCTCAGG 137854474 NM_000138.4(FBN1):c.3793T>C CTTGYGTTATGATGGATTCATGG Marfan syndrome (p.Cys1265Arg) 587784418 NM_006306.3(SMC1A):c.3254A>G CTTAYAGATCTCATCAATGTTGG Congenital muscular hypertrophy-cerebral (p.Tyr1085Cys) syndrome 81002805 NM_000059.3(BRCA2):c.316+2T>C CTTAGGYAAGTAATGCAATATGG Familial cancer of breast, Breast-ovarian cancer, familial 2, Hereditary cancer-predisposing syndrome 121909653 NM_182925.4(FLT4):c.3104A>G CTGYGGATGCACTGGGGTGCGGG, (p.His1035Arg) TCTGYGGATGCACTGGGGTGCGG 786205107 NM_031226.2(CYP19A1):c.743+2T>C CTGTGYAAGTAATACAACTTTGG Aromatase deficiency 587777037 NM_001283009.1(RTEL1):c.3730T>C CTGTGTGYGCCAGGGCTGTGGGG Dyskeratosis congenita, autosomal recessive, 5 (p.Cys1244Arg) 794728380 NM_000238.3(KCNH2):c.1945+6T>C CTGTGAGYGTGCCCAGGGGCGGG, Cardiac arrhythmia TGAGYGTGCCCAGGGGCGGGCGG 267607987 NM_000251.2(MSH2):c.2005+2T>C CTGGYAAAAAACCTGGTTTTTGG, Hereditary Nonpolyposis Colorectal Neoplasms TGGYAAAAAACCTGGTTTTTGG G 397509397 NM_006876.2(B4GAT1):c.1168A>G TGATYTTCAGCCTCCTTTTGGGG, Congenital muscular dystrophy-dystroglycanopathy (p.Asn390Asp) CTGATYTTCAGCCTCCTTTTGGG, with brain and eye anomalies, type A13 GCTGATYTTCAGCCTCCTTTTGG 121918381 NM_000040.1(APOC3):c.280A>G CTGAAGYTGGTCTGACCTCAGGG, (p.Thr94Ala) GCTGAAGYTGGTCTGACCTCAGG 104894919 NM_001015877.1(PHF6):c.769A>G CTCYTGATGTTGTTGTGAGCTGG Borjeson-Forssman-Lehmann syndrome (p.Arg257Gly) 267606869 NM_005144.4(HR):c.-218A>G CTCYAGGGCCGCAGGTTGGAGGG, Marie Unna hereditary hypotrichosis 1 GCTCYAGGGCCGCAGGTTGGAGG, GGCGCTCYAGGGCCGCAGGTTGG 139732572 NM_000146.3(FTL):c.1A>G CTCAYGGTTGGTTGGCAAGAAGG L-ferritin deficiency (p.Met1Val) 397515418 NM_018486.2(HDAC8):c.1001A>G CTCAYGATCTGGGATCTCAGAGG Cornelia de Lange syndrome 5 (p.His334Arg) 372395294 NM_198056.2(SCN5A):c.1247A>G CTCAYAGGCCATTGCGACCACGG not provided (p.Tyr416Cys) 104895304 NM_000431.3(MVK):c.803T>C CTCAAYAGATGCCATCTCCCTGG Hyperimmunoglobulin D with periodic (p.Ile26811r) fever, Mevalonic aciduria 587777188 NM_001165899.1(PDE4D):c.1850T>C CTATAYTGTTCATCCCCTCTGGG, Acrodysostosis 2, with or without hormone (p.Ile617Thr) ACTATAYTGTTCATCCCCTCTGG resistance 398123026 NM_003867.3(FGF17):c.560A>G CGTGGYTGGGGAAGGGCAGCTGG Hypogonadotropic hypogonadism 20 with or (p.Asn187Ser) without anosmia 121964924 NM_001385.2(DPYS):c.1078T>C CGTAATAYGGGAAAAAGGCGTGG, Dihydropyrimidinase deficiency (p.Trp360Arg) AATAYGGGAAAAAGGCGTGGTGG, ATAYGGGAAAAAGGCGTGGTGGG 587777301 NM_199189.2(MATR3):c.1864A>G CGGYTGAACTCTCAGTCTTCTGG Myopathy, distal, 2 (p.Thr622Ala) 200238879 NM_000527.4(LDLR):c.694+2T>C ACTGCGGYATGGGCGGGGCCAGG, Familial hypercholesterolemia CTGCGGYATGGGCGGGGCCAGGG, CGGYATGGGCGGGGCCAGGGTGG 142951029 NM_145046.4(CALR3):c.245A>G CGGTYTGAAGCGTGCAGAGATGG Arrhythmogenic right ventricular (p.Lys82Arg) cardiomyopathy, Familial hypertrophic cardiomyopathy 19, Hypertrophic cardiomyopathy 786200953 NM_006785.3(MALTI):c.1019- CGCYTTGAAAAAAAAAGAAAGGG, Combined immunodeficiency

TCGCYTTGAAAAAAAAAGAAAG 120074192 NM_000218.2(KCNQ1):c.418A>G CGCYGAAGATGAGGCAGACCAGG Atrial fibrillation, familial, 3, Atrial fibrillation (p.Ser140Gly) 267606887 NM_005957.4(MTHFR):c.971A>G CGCGGYTGAGGGTGTAGAAGTGG Homocystinuria due to MTHFR deficiency (p.Asn324Ser) 118192117 NM_000540.2(RYR1):c.1205T>C CGCAYGATCCACAGCACCAATGG Congenital myopathy with fiber (p.Met402Thr) type disproportion, Central core disease 199473625 NM_198056.2(SCN5A):c.4978A>G CGAYGTTGAAGAGGGCAGGCAGG, Brugada syndrome (p.Ile1660Val) AGCCCGAYGTTGAAGAGGGCAGG 794726865 NM_000921.4(PDE3A):c.1333A>G CGAGGYGGTGGTGGTCCAAGTGG Brachydactyly with hypertension (p.Thr445Ala) 606231254 NM_005740.2(DNAL4):c.153+2T>C CGAGGYATTGCCAGCAGTGCAGG Mirror movements 3 786204826 NM_004771.3(MMP20):c.611A>G CGAAAYGTGTATCTCCTCCCAGG Amelogenesis imperfecta, hypomaturation type, (p.His204Arg) IIA2 796053139 NM_021007.2(SCN2A):c.4308+2T>C CGAAATGYAAGTCTAGTTAGAGG, not provided GAAATGYAAGTCTAGTTAGAGG 137854494 NM_005502.3(ABCA1):c.4429T>C CCTGTGYGTCCCCCAGGGGCAGG, Tangier disease (p.Cys1477Arg) CTGTGYGTCCCCCAGGGGCAGGG, TGTGYGTCCCCCAGGGGCAGGGG, GTGYGTCCCCCAGGGGCAGGGGG 786205144 NM_001103.3(ACTN2):c.683T>C CCTAAAAYGTTGGATGCTGAAGG Dilated cardiomyopathy IAA (p.Met228Thr) 199919568 NM_007254.3(PNKP):c.1029+2T>C CCGGYGAGGCCCTGGGGCGGGGG, not provided TCCGGYGAGGCCCTGGGGCGGGG, ATCCGGYGAGGCCCTGGGGCGGG, GATCCGGYGAGGCCCTGGGGCGG 28939079 NM_018965.3(TREM2):c.401A>G TGAYCCAGGGGGTCTATGGGAGG, Polycystic lipomembranous osteodysplasia with (p.Asp134Gly) CGGTGAYCCAGGGGGTCTATGGG, sclerosing leukoencephalopathy CCGGTGAYCCAGGGGGTCTATGG 193302855 NM_032520.4(GNPTG):c.610-2A>G CCCYGAAGGTGGAGGATGCAGGG, Mucolipidosis III Gamma GCCCYGAAGGTGGAGGATGCAGG 111033708 NM_000155.3(GALT):c.499T>C CCCTYGGGTGCAGGTTTGTGAGG Deficiency of UDPglucose-hexose-1-phosphate (p.Trp167Arg) uridylyltransferase 28933378 NM_000174.4(GP9):c.70T>C CCCAYGTACCTGCCGCGCCCTGG Bernard Soulier syndrome, Bernard-Soulier (p.Cys24Arg) syndrome type C 364897 NM_000157.3(GBA):c.680A>G CCAYTGGTCTTGAGCCAAGTGGG, Gaucher disease, Subacute neuronopathic (p.Asn227Ser) TCCAYTGGTCTTGAGCCAAGTGG Gaucher disease, Gaucher disease, type 1 796052551 NM_000833.4(GRIN2A):c.2449A>G CCAYGTTGTCAATGTCCAGCTGG not provided (p.Met817Val) 63751006 NM_002087.3(GRN):c.2T>C CCAYGTGGACCCTGGTGAGCTGG Frontotemporal dementia, ubiquitin-positive (p.Met1Thr) 786203997 NM_001031.4(RPS28):c.1A>G TGTCCAYGATGGCGGCGCGGCGG, Diamond-Blackfan anemia with microtia and (p.Met1Val) CCAYGATGGCGGCGCGGCGGCGG cleft palate 121908595 NM_002755.3(MAP2K1):c.389A>G CCAYAGAAGCCCACGATGTACGG Cardiofaciocutaneous syndrome 3, Rasopathy (p.Tyr130Cys) 398122910 NM_000431.3(MVK):c.1039+2T>C CCAGGYATCCCGGGGGTAGGTGG, Porokeratosis, disseminated superficial actinic CAGGYATCCCGGGGGTAGGTGGG 1 119474039 NM_020365.4(EIF2B3):c.1037T>C CCAGAYTGTCAGCAAACACCTGG Leukoencephalopathy with vanishing white (p.Ile346Thr) matter 587777866 NM_000076.2(CDKN1C):c.*5+2T>C CCAAGYGAGTACAGCGCACCTGG, Beckwith-Wiedemann syndrome CAAGYGAGTACAGCGCACCTGGG, AAGYGAGTACAGCGCACCTGGGG 121918530 NM_005587.2(MEF2A):c.788A>G AGAYTACCACCACCTGGTGGAGG, (p.Asn263Ser) CCAAGAYTACCACCACCTGGTGG 483352818 NM_000211.4(ITGB2):c.1877+2T>C CATGYGAGTGCAGGCGGAGCAGG Leukocyte adhesion deficiency type 1 460184 NM_000186.3(CFH):c.3590T>C CAGYTGAATTTGTGTGTAAACGG Atypical hemolytic-uremic syndrome 1 (p.Val1197Ala) 121908423 NM_004795.3(KL):c.578A>G CAGYGGTACAGGGTGACCACGGG, (p.His193Arg) CCAGYGGTACAGGGTGACCACGG 281860300 NM_005247.2(FGF3):c.146A>G CAGYAGAGCTTGCGGCGCCGGGG, Deafness with labyrinthine aplasia microtia and (p.Tyr49Cys) GCAGYAGAGCTTGCGGCGCCGGG, microdontia (LAIVIM) CGCAGYAGAGCTTGCGGCGCCGG 28935488 NM_000169.2(GLA):c.806T>C CAGTTAGYGATTGGCAACTTTGG Fabry disease (p.Val269Ala) 587776514 NM_173560.3(RFX6):c.380+2T>C CAGTGGYGAGACTCGCCCGCAGG, Mitchell-Riley syndrome AGTGGYGAGACTCGCCCGCAGGG 104894117 NM_178138.4(LHX3):c.332A>G CAGGTGGYACACGAAGTCCTGGG Pituitary hormone deficiency, combined 3 (p.Tyr111Cys) 34878913 NM_000184.2(HBG2):c.125T>C CAGAGGTYCTTTGACAGCTTTGG Cyanosis, transient neonatal (p.Phe42Ser) 120074124 NM_000543.4(SMPD1):c.911T>C AGCACYTGTGAGGAAGTTCCTGG, Sphingomyelin/cholesterol lipidosis,Niemann- (p.Leu304Pro) GCACYTGTGAGGAAGTTCCTGGG, Pick disease, type A, Niemann-Pick disease, CACYTGTGAGGAAGTTCCTGGGG type B 281860272 NM_005211.3(CSF1R):c.2320-2A>G CACYGAGGGAAAGCACTGCAGGG, Hereditary diffuse leukoencephalopathy with GCACYGAGGGAAAGCACTGCAGG spheroids 128624216 NM_000033.3(ABCD1):c.443A>G CACTGYTGACGAAGGTAGCAGGG, Adrenoleukodystrophy (p.Asn148Ser) GCACTGYTGACGAAGGTAGCAGG 398124257 NM_012463.3(ATP6V0A2):c.825+2 CACTGYGAGTAAGCTGGAAGTGG Cutis laxa with osteodystrophy T>C 267606679 NM_004183.3(BEST I):c.704T>C CACTGGYGTATACACAGGTGAGG Vitreoretinochoroidopathy dominant (p.Val235Ala) 397514518 NM_000344.3(SMNI):c.388T>C CACTGGAYATGGAAATAGAGAGG Kugelberg-Welander disease (p.Tyr130His) 143946794 NM_001946.3(DUSP6):c.566A>G CACTAYTGGGGTCTCGGTCAAGG Hypogonadotropic hypogonadism 19 with or (p.Asn189Ser) without anosmia 397516076 NM_000256.3(MYBPC3):c.821+2T GCACGYGAGTGGCCATCCTCAGG, Familial hypertrophic cardiomyopathy 4, not

CACGYGAGTGGCCATCCTCAGGG specified 149977726 NM_001257988.1(TYMP):c.665A>G CACGAGTYTCTTACTGAGAATGG, (p.Lys222Arg) GAGTYTCTTACTGAGAATGGAGG 121917770 NM_003361.3(UMOD):c.383A>G CACAYTGACACATGTGGCCAGGG, Familial juvenile gout (p.Asn128Ser) CCACAYTGACACATGTGGCCAGG 121909008 NM_000492.3(CFTR):c.2738A>G CACATAAYACGAACTGGTGCTGG Cystic fibrosis (p.Tyr913Cys) 137852819 NM_003688.3(CASK):c.2740T>C CACAGYGGGTCCCTGTCTCCTGG, FG syndrome 4 (p.Trp914Arg) ACAGYGGGTCCCTGTCTCCTGGG 74315320 NM_024009.2(GM3):c.421A>G CAAYGATGAGCTTGAAGATGAGG Deafness, autosomal recessive (p.Ile141Val) 80356747 NM_001701.3(BAAT):c.967A>G CAAYGAAGAGGAATTGCCCCTGG Atypical hemolytic-uremic syndrome 1 (p.Ile323Val) 180177324 NM_012203.1(GRHPR):c.934A>G CAAGTYGTTAGCTGCCAACAAGG Primary hyperoxaluria, type II (p.Asn312Asp) 281860274 NM 005211.3(CSFIR):c.238IT>C CAAGAYTGGGGACTTCGGGCTGG Hereditary diffuse leukoencephalopathy with (p.Ile794Thr) spheroids 398122908 NM_005334.2(HCFC1):c.- CAAGAYGGCGGCTCCCAGGGAGG Mental retardation 3, X-linked 970T>C 548076633 NM_002693.2(POLG):c.3470A>G CAAGAGGYTGGTGATCTGCAAGG not provided (p.Asn1157Ser) 120074146 NM_000019.3(ACAT1):c.935T>C CAAGAAYAGTAGGTAAGGCCAGG Deficiency of acetyl-CoA acetyltransferase (p.Ile312Thr) 397514489 NM_005340.6(HINT1):c.250T>C CAAGAAAYGTGCTGCTGATCTGG, Gamstorp-Wohlfart syndrome (p.Cys84Arg) AAGAAAYGTGCTGCTGATCTGGG 587783539 NM_178151.2(DCX):c.2T>C CAAAATAYGGAACTTGATTTTGG Heterotopia (p.Met1Thr) 104894765 NM_005448.2(BMP15):c.704A>G ATTGAAAYAGAGTAACAAGAAGG Ovarian dysgenesis 2 (p.Tyr235Cys) 137852429 NM_000132.3(F8):c.1892A>G ATGYTGGAGGCTTGGAACTCTGG Hereditary factor VIII deficiency disease (p.Asn631Ser) 72558441 NM_000531.5(OTC):c.779T>C ATGTATYAATTACAGACACTTGG not provided (p.Leu260Ser) 398123765 NM_003494.3(DYSF):c.1284+2T>C ATGGYAAGGAGCAAGGGAGCAGG Limb-girdle muscular dystrophy, type 2B 387906924 NM_020191.2(MRPS22):c.644T>C ATCYTAGGGTAAGGTGACTTAGG Combined oxidative phosphorylation deficiency (p.Leu215Pro) 5 397518039 NM_206933.2(USH2A):c.8559- ATCYAAAGCAAAAGACAAGCAGG Retinitis pigmentosa, Usher syndrome, type 2A 2A>G 5742905 NM_000071.2(CBS):c.833T>C ATCAYTGGGGTGGATCCCGAAGG, Homocystinuria due to CBS (p.Ile278Thr) TCAYTGGGGTGGATCCCGAAGGG deficiency, Homocystinuria,

397507473 NM_004333.4(BRAF):c.403T>C ATCATYTGGAACAGTCTACAAGG, Cardiofaciocutaneous syndrome, Rasopathy (p.Phe468Ser) TCATYTGGAACAGTCTACAAGG 786204056 NM_000264.3(PTCHH:c.3168+2T>C ATCATTGYGAGTGTATTATAAGG, Gorlin syndrome TCATTGYGAGTGTATTATAAGGG, CATTGYGAGTGTATTATAAGGG 72558484 NM_000531.5(OTC):c.1005+2T>C ATCATGGYAAGCAAGAAACAAGG not provided 199473074 NM_000335.4(SCN5A):c.688A>G ATAYAGTTTTCAGGGCCCGGAGG, Brugada syndrome (p.Ile230Val) CTGATAYAGTTTTCAGGGCCCGG 111033273 NM_206933.2(USH2A):c.1606T>C ATATAGAYGCCTCTGCTCCCAGG Usher syndrome, type 2A (p.Cys536Arg) 72556290 NM_000531.5(OTC):c.542A>G ATAGTGTYCCTAAAAGGCACGGG not provided (p.Glu181Gly) 121918711 NM_004612.3(TGFBR1):c.1199A>G ATAGATGYCAGCACGTTTGAAGG Loeys-Dietz syndrome 1 (p.Asp400Gly) 104886288 NM_000495.4(COL4A5):c.4699T>C AGTAYGTGAAGCTCCAGCTGTGG Alport syndrome, X-linked recessive (p.Cys1567Arg) 144637717 NM_016725.2(FOLRH:c.493+2T>C CTTCAGGYGAGGGCTGGGGTGGG, not provided AGGYGAGGGCTGGGGTGGGCAGG 72558492 NM_000531.5(OTC):c.1034A>G AGGTGAGYAATCTGTCAGCAGGG not provided (p.Tyr345Cys) 62638745 NM_000121.3(EPOR):c.1460A>G AGGGYTGGAGTAGGGGCCATCGG Acute myeloid leukemia, M6 type, (p.Asn487Ser) Familial erythrocytosis, 1 387907021 NM_031427.3(DNAL1):c.449A>G AGGGAYTGCCTACAAACACCAGG Kartagener syndrome, Ciliary dyskinesia, (p.Asn150Ser) primary, 16 397514488 NM_001161581.1(POC1A):c.398T>C AGCYGTGGGACAAGAGCAGCCGG Short stature, onychodysplasia, facial (p.Leu133Pro) dysmorphism, and hypotrichosis 154774633 NM_017882.2(CLN6):c.200T>C AGCYGGTATTCCCTCTCGAGTGG Adult neuronal ceroid lipofuscinosis (p.Leu67Pro) 111033700 NM_000155.3(GALT):c.482T>C AGCYGGGTGCCCAGTACCCTTGG Deficiency of UDPglucose-hexose-1-phosphate (p.Leu161Pro) uridylyltransferase 128621198 NM_000061.2(BTK):c.1223T>C GAGCYGGGGACTGGACAATTTGG, X-linked agammaglobulinemia (p.Leu408Pro) AGCYGGGGACTGGACAATTTGGG 137852611 NM_000211.4(ITGB2):c.446T>C AGCYAGGTGGCGACCTGCTCCGG Leukocyte adhesion deficiency (p.Leu149Pro) 121908838 NM_003722.4(TP63):c.697A>G AGCTTYTTTGTAGACAGGCATGG Split-hand/foot malformation 4 (p.Lys233Glu) 397515869 NM_000169.2(GLA):c.1153A>G AGCTGTGYGATGAAGCAGGCAGG not specified (p.Thr385Ala) 118204064 NM_000237.2(LPL):c.548A>G GCTGGAYCGAGGCCTTAAAAGGG, Hyperlipoproteinemia, type I (p.Asp183Gly) AGCTGGAYCGAGGCCTTAAAAGG 128620186 NM_000061.2(BTK):c.2T>C AGCTAYGGCCGCAGTGATTCTGG X-linked agammaglobulinemia (p.Met1Thr) 786204132 NM_014946.3(SPAST):c.1165A>G ATTGYCTTCCCATTCCCAGGTGG, Spastic paraplegia 4, autosomal dominant (p.Thr389Ala) AGCATTGYCTTCCCATTCCCAGG 199473661 NM_000218.2(KCNQ1):c.550T>C CAGCAAGBACGTGGGCCTCTGGG, Congenital long QT syndrome, Cardiac (p.Tyr184His) AGCAAGBACGTGGGCCTCTGGGG, arrhythmia GCAAGBACGTGGGCCTCTGGGGG 387907129 NM_024599.5(RHBDF2):c.557T>C AGAYTGTGGATCCGCTGGCCCGG Howel-Evans syndrome (p.Ile186Thr) 387906702 NM_006306.3(SMC1A):c.2351T>C AGAYTGGTGTGCGCAACATCCGG Congenital muscular hypertrophy-cerebral (p.Ile784Thr) syndrome 193929348 NM_000525.3(KCNJ11):c.544A>G AGAYGAGGGTCTCAGCCCTGCGG Permanent neonatal diabetes mellitus (p.Ile182Val) 121908934 NM_004086.2(COCH):c.1535T>C AGATAYGGCTTCTAAACCGAAGG Deafness, autosomal dominant 9 (p.Met512Thr) 397514377 NM_000060.3(BTD):c.641A>G AGAGGYTGTGTTTACGGTAGCGG Biotinidase deficiency (p.Asn214Ser) 72552295 NM_000531.5(OTC):c.2T>C AGAAGAYGCTGTTTAATCTGAGG not provided (p.Met1Thr) 201893545 NM_016247.3(IMPG2):c.370T>C ACTYTTTGGGATCGACTTCCTGG Macular dystrophy, vitelliform, 5 (p.Phe124Leu) 121434469 m.4290T>C ACTYTGATAGAGTAAATAATAGG 121918733 NM_006920.4(SCN1A):c.269T>C ACTTYTATAGTATTGAATAAAGG, Severe myoclonic epilepsy in infancy (p.Phe90Ser) CTTYTATAGTATTGAATAAAGG G 121434471 m.4291T>C ACTTYGATAGAGTAAATAATAGG Hypertension, hypercholesterolemia, and hypomagnesemia, mitochondrial 606231289 NM_001302946.1(TRNT1):c.497T>C ACTTYATTTGACTACTTTAATGG Sideroblastic anemia with B-cell (p.Leu166Ser) immunodeficiency, periodic fevers, and developmental delay 63750067 NM_000517.4(HBA2):c.*92A>G CTTYATTCAAAGACCAGGAAGGG, Hemoglobin H disease, nondeletional ACTTYATTCAAAGACCAGGAAG G 121918734 NM_006920.4(SCN1A):c.272T>C ACTTTTAYAGTATTGAATAAAGG, Severe myoclonic epilepsy in infancy (p.Ile91Thr) CTTTTAYAGTATTGAATAAAGG G 137854557 NM_000267.3(NF1):c.1466A>G ACTTAYAGCTTCTTGTCTCCAGG Neurofibromatosis, type 1 (p.Tyr489Cys) 397514626 NM_018344.5(SLC29A3):c.607T>C ACTGATAYCAGGTGAGAGCCAGG, Histiocytosis-lymphadenopathy plus syndrome (p.Ser203Pro) CTGATAYCAGGTGAGAGCCAGGG 118204440 NM_000512.4(GALNS):c.1460A>G ACGYTGAGCTGGGGCTGCGCGGG, Mucopolysaccharidosis, MPS-IV-A (p.Asn487Ser) CACGYTGAGCTGGGGCTGCGCGG 587776843 NG_012088.1:g.2209A>G ACCYTATGATCCGCCCGCCTTGG 137853033 NM_001080463.1(DYNC2H1):c.4610A>G ACCYGTGAAGGGAACAGAGATGG Short-rib thoracic dysplasia 3 with or (p.Gln1537Arg) without polydactyly 28933698 NM_000435.2(NOTCH3):c.1363T>C TTCACCYGTATCTGTATGGCAGG, Cerebral autosomal dominant arteriopathy with (p.Cys455Arg) ACCYGTATCTGTATGGCAGGTGG subcortical infarcts and leukoencephalopathy ACCYGAGATGCAAAATAGGGAGG, 587776766 NM_000463.2(UGT1A1):c.1085-2A>G GTGACCYGAGATGCAAAATAGGG, Crigler Najjar syndrome, type 1 GGTGACCYGAGATGCAAAATAGG 587781628 NM_001128425.1(MUTYH):c.1187-2A>G ACCYGAGAGGGAGGGCAGCCAGG Hereditary cancer-predisposing syndrome, Carcinoma of colon 61755817 NM_000322.4(PRPH2):c.736T>C ACCTGYGGGTGCGTGGCTGCAGG, Retinitis pigmentosa (p.Trp246Arg) CCTGYGGGTGCGTGGCTGCAGGG 121909184 NM_001089.2(ABCA3):c.1702A>G ACCGTYGTGGCCCAGCAGGACGG Surfactant metabolism dysfunction, pulmonary, (p.Asn568Asp) 3 121434466 m.4269A>G ACAYATTTCTTAGGTTTGAGGGG, GACAYATTTCTTAGGTTTGAGGG, AGACAYATTTCTTAGGTTTGAGG 794726768 NM_001165963.1(SCN1A):c.1048A>G ACAYATATCCCTCTGGACATTGG Severe myoclonic epilepsy in infancy (p.Met350Val) 28934876 NM_001382.3(DPAGT1):c.509A>G ACAYAGTACAGGATTCCTGCGGG, Congenital disorder of glycosylation type 1J (p.Tyr170Cys) GACAYAGTACAGGATTCCTGCGG 104894749 NM_000054.4(AVPR2):c.614A>G ACAYAGGTGCGACGGCCCCAGGG, Nephrogenic diabetes insipidus, Nephrogenic (p.Tyr205Cys) GACAYAGGTGCGACGGCCCCAGG diabetes insipidus, X-linked 128621205 NM_000061.2(BTK):c.1741T>C ACATTYGGGCTTTTGGTAAGTGG X-linked agammaglobulinemia (p.Trp581Arg) 28940892 NM_000529.2(MC2R):c.761A>G ACATGYAGCAGGCGCAGTAGGGG, ACTH resistance (p.Tyr254Cys) GACATGYAGCAGGCGCAGTAGGG, AGACATGYAGCAGGCGCAGTAGG 794726844 NM_001165963.1(SCN1A):c.1046A>G ACATAYATCCCTCTGGACATTGG Severe myoclonic epilepsy in infancy (p.Tyr349Cys) 587783083 NM_003159.2(CDKL5):c.449A>G ACAGTYTTAGGACATCATTGTGG not provided (p.Lys150Arg) 397514651 NM_000108.4(DLD):c.140T>C ACAGTTAYAGGTTCTGGTCCTGG, Maple syrup urine disease, type 3 (p.Ile47Thr) GTTAYAGGTTCTGGTCCTGGAGG 794727060 NM_001848.2(COL6A1):c.957+2T>C ACAAGGYGAGCGTGGGCTGCTGG, Ullrich congenital muscular dystrophy, Bethlem CAAGGYGAGCGTGGGCTGCTGGG myopathy 72554346 NM_000531.5(OTC):c.284T>C ACAAGATYGTCTACAGAAACAGG not provided (p.Leu95Ser) 483353031 NM_002136.2(HNRNPA1):c.841T>C AATYTTGGAGGCAGAAGCTCTGG Chronic progressive multiple sclerosis (p.Phe281Leu) 104894271 NM_000315.2(PTH):c.52T>C AATTYGTTTTCTTACAAAATCGG Hypoparathyroidism familial isolated (p.Cys18Arg) 267608260 NM_015599.2(PGM3):c.248T>C AATGTYGGCACCATCCTGGGAGG Immunodeficiency 23 (p.Leu83Ser) 267606900 NM_018109.3(MTPAP):c.1432A>G AATGGATYCTGAATGTACAGAGG Ataxia, spastic, 4, autosomal recessive (p.Asn478Asp) 796053169 NM_021007.2(SCN2A):c.387- AATAAAGYAGAATATCGTCAAGG not provided 2A>G 104894937 NM_000116.4(TAZ):c.352T>C AAGYGTGTGCCTGTGTGCCGAGG 3-Methylglutaconic aciduria type 2 (p.Cys118Arg) 104893911 NM_001018077.1(NR3C1):c.1712T>C AAGYGATTGCAGCAGTGAAATGG Pseudohermaphroditism, female, with (p.Val571Ala) hypokalemia, due to glucocorticoid resistance 397514472 NM_004813.2(PEX16):c.992A>G AAGYAGATTTTCTGCCAGGTGGG, Peroxisome biogenesis disorder 8B (p.Tyr331Cys) GAAGYAGATTTTCTGCCAGGTGG, GTAGAAGYAGATTTTCTGCCAGG 121918407 NM_001083112.2(GPD2):c.1904T>C AAGTYTGATGCAGACCAGAAAGG Diabetes mellitus type 2 (p.Phe635Ser) 63751110 NM_000251.2(MSH2):c.595T>C AAGGAAYGTGTTTTACCCGGAGG Hereditary Nonpolyposis Colorectal Neoplasms (p.Cys199Arg) 119450945 NM_000026.2(ADSL):c.674T>C AAGAYGGTGACAGAAAAGGCAGG Adenylosuccinate lyase deficiency (p.Met225Thr) 113993988 NM_002863.4(PYGL):c.2461T>C AAGAAYATGCCCAAAACATCTGG Glycogen storage disease, type VI (p.Tyr821His) 119485091 NM_022041.3(GAN):c.1268T>C AAGAAAAYCTACGCCATGGGTGG, Giant axonal neuropathy (p.Ile423Thr) AAAAYCTACGCCATGGGTGGAGG 137852419 NM_000132.3(F8):c.1660A>G AACYAGAGTAATAGCGGGTCAGG Hereditary factor VIII deficiency disease (p.Ser554Gly) 121964967 NM_000071.2(CBS):c.1150A>G AACTYGGTCCTGCGGGATGGGGG, Homocystinuria,pyridox ne-respons ve (p.Lys384Glu) GAACTYGGTCCTGCGGGATGGGG, GGAACTYGGTCCTGCGGGATGGG, AGGAACTYGGTCCTGCGGGATGG 137852376 NM_000132.3(F8):c.1754T>C AACAGAYAATGTCAGACAAGAGG Hereditary factor VIII deficiency disease (p.Ile585Thr) 121917930 NM_006920.4(SCN1A):c.3577T>C AACAAYGGTGGAACCTGAGAAGG Generalized epilepsy with febrile seizures plus, (p.Trp1193Arg) type 1, Generalized epilepsy with febrile seizures plus, type 2 28939717 NM_003907.2(EIF2B5):c.271A>G AAATGYTTCCTGTACACCTGTGG Leukoencephalopathy with vanishing white (p.Thr91Ala) matter 80357276 NM_007294.3(BRCA1):c.122A>G AAATATGYGGTCACACTTTGTGG Familial cancer of breast, Breast-ovarian (p.His41Arg) cancer, familial 1 397515897 NM_000256.3(MYBPC3):c.1351+2T>C AAAGGYGGGCCTGGGACCTGAGG Familial hypertrophic cardiomyopathy 4, Cardiomyopathy 397514491 NM_005340.6(HINT I):c.152A>G AAAAYGTGTTGGTGCTTGAGGGG, Gamstorp-Wohlfart syndrome (p.His5lArg) GAAAAYGTGTTGGTGCTTGAGGG, AGAAAAYGTGTTGGTGCTTGAGG 387907164 NM_020894.2(UVSSA):c.94T>C AAAATTYGCAAGTATGTCTTAGG, UV-sensitive syndrome 3 (p.Cys32Arg) AAATTYGCAAGTATGTCTTAGG G 118161496 NM_025152.2(NUBPL):c.815- TGGTTCYAATGGATGTCTGCTGG, Mitochondrial complex I deficiency 27T>C GGTTCYAATGGATGTCTGCTGGG 764313717 NM_005609.2(PYGM):c.425_528del TGGCTGYCAGGGACCCAGCAAGG, CTGYCAGGGACCCAGCAAGGAGG 28934568 NM_003242.5(TGFBR2):c.923T>C AGTTCCYGACGGCTGAGGAGCGG Loeys-Dietz syndrome 2 (p.Leu308Pro) 121913461 NM_007313.2(ABL1):c.814T>C CCAGYACGGGGAGGTGTACGAGG, (p.Tyr272His) CAGYACGGGGAGGTGTACGAGGG 377750405 NM_173551.4(ANKS6):c.1322A>G AGGGCYGTCGGACCTTCGAGTGG, Nephronophthisis 16 (p.Gln441Arg) GGGCYGTCGGACCTTCGAGTGGG, GGCYGTCGGACCTTCGAGTGGGG 57639980 NM_001927.3(DES):c.1034T>C ATTCCCYGATGAGGCAGATGCGG, Myofibrillar myopathy 1 (p.Leu345Pro) TTCCCYGATGAGGCAGATGCGGG 147391618 NM_020320.3(RARS2):c.35A>G ATACCYGGCAAGCAATAGCGCGG Pontocerebellar hypoplasia type 6 (p.Gln12Arg) 182650126 NM_002977.3(SCN9A):c.2215A>G GTAAYTGCAAGATCTACAAAAGG Small fiber neuropathy (p.Ile739Val) 80358278 NM_004700.3(KCNQ4):c.842T>C ACATYGACAACCATCGGCTATGG DFNA 2 Nonsyndromic Hearing Loss (p.Leu281Ser) 786204012 NM_005957.4(MTHFR):c.388T>C GACCYGCTGCCGTCAGCGCCTGG Homocysteinemia due to MTHFR deficiency (p.Cys130Arg) 786204037 NM_005957.4(MTHFR):c.1883T>C TCCCACYGGACAACTGCCTCTGG Homocysteinemia due to MTHFR deficiency (p.Leu628Pro) 202147607 NM_000140.3(FECH):c.1137+3A>G GTAGAYACCTTAGAGAACAATGG Erythropoieticprotoporphyria 122456136 NM_005183.3(CACNA1F):c.2267T>C TGCCAYTGCTGTGGACAACCTGG (p.Ile756Thr) 786204851 NM_007374.2(SIX6):c.110T>C GTCGCYGCCCGTGGCCCCTGCGG Cataract, microphthalmia and nystagmus (p.Leu37Pro) 794728167 NM_000138.4(FBN1):c.1468+2T>C ATTGGYACGTGATCCATCCTAGG Thoracic aortic aneurysms and aortic dissections 121964909 NM_000027.3(AGA):c.214T>C GACGGCYCTGTAGGCTTTGGAGG Aspartylglycosaminuria (p.Ser72Pro) 121964978 NM_000170.2(GLDC):c.2T>C CGGCCAYGCAGTCCTGTGCCAGG, Non-ketotichyperglycinemia (p.Met1Thr) GGCCAYGCAGTCCTGTGCCAGGG 121965008 NM_000398.6(CYB5R3):c.446T>C CTGCYGGTCTACCAGGGCAAAGG METHEMOGLOBINEMIA, TYPE I (p.Leu149Pro) 121965064 NM_000128.3(F1 1 ):c.90IT>C TGATYTCTTGGGAGAAGAACTGG Hereditary factor XI deficiency disease (p.Phe301Leu) 45517398 NM_000548.3(TSC2):c.5150T>C GCCCYGCACGCAAATGTGAGTGG, Tuberous sclerosis syndrome (p.Leu1717Pro) CCCYGCACGCAAATGTGAGTGGG 786205857 NM_015662.2(IFT172):c.770T>C TTGTGCYAGGAAGTTATGACAGG RETINITIS PIGMENTOSA 71 (p.Leu257Pro) 786205904 NM_001135669.1(XPR1):c.653T>C GCGTTYACGTGTCCCCCCTTTGG, BASAL GANGLIA (p.Leu218Ser) CGTTYACGTGTCCCCCCTTTGGG CALCIFICATION, 104893704 NM_000388.3(CASR):c.2641T>C ACGCTYTCAAGGTGGCTGCCCGG, Hypercalciuric hypercalcemia (p.Phe881Leu) CGCTYTCAAGGTGGCTGCCCGGG 104893747 NM_I98159.2(MITF):c.1195T>C ACTTYCCCTTATTCCATCCACGG, Waardenburg syndrome type 2A (p.Ser399Pro) CTTYCCCTTATTCCATCCACGGG 104893770 NM_000539.3(RHO):c.133T>C CATGYTTCTGCTGATCGTGCTGG, Retinitis pigmentosa 4 (p.Phe45Leu) ATGYTTCTGCTGATCGTGCTGGG 28937596 NM_003907.2(EIF2B5):c.1882T>C AGGCCYGGAGCCCTGTTTTTAGG Leukoencephalopathy with vanishing white (p.Trp628Arg) matter 104893876 NM_001151.3(SLC25A4):c.293T>C GCAGCYCTTCTTAGGGGGTGTGG Autosomal dominant progressive external (p.Leu98Pro) ophthalmoplegia with mitochondrial DNA deletions 2 104893883 NM_006005.3(WFS1):c.2486T>C ACCATCCYGGAGGGCCGCCTGGG WFS1-Related Disorders (p.Leu829Pro) 104893962 NM_000165.4(GJA1):c.52T>C CTACYCAACTGCTGGAGGGAAGG Oculodentodigital dysplasia (p.Ser18Pro) 104893978 NM_000434.3(NEU1):c.718T>C GCCTCCYGGCGCTACGGAAGTGG, Sialidosis, type II (p.Trp240Arg) CCTCCYGGCGCTACGGAAGTGGG, CTCCYGGCGCTACGGAAGTGGGG 104894092 NM_002546.3(TNFRSF11B):c.349T>C TAGAGYTCTGCTTGAAACATAGG Hyperphosphatasemia with bone disease (p.Phe117Leu) 104894135 NM_000102.3(CYP17A1):c.316T>C CATCGCGYCCAACAACCGTAAGG, Complete combined 17-alpha- (p.Ser106Pro) ATCGCGYCCAACAACCGTAAGGG hydroxylase/17,20-lyase 104894151 NM_000102.3(CYP17A1):c.1358T>C AGCTCTYCCTCATCATGGCCTGG Combined partial 17-alpha-hydroxylase/17,20- (p.Phe453Ser) lyase deficiency 36015961 NM_000518.4(HBB):c.344T>C TGTGTGCYGGCCCATCACTTTGG Beta thalassemia intermedia (p.Leu115Pro) 104894472 NM_152443.2(RDH12):c.523T>C TCCYCGGTGGCTCACCACATTGG Leber congenital amaurosis 13 (p.Ser175Pro) 104894587 NM_004870.3(MPDU1):c.356T>C TTCCYGGTCATGCACTACAGAGG Congenital disorder of glycosylation type 1F (p.Leu119Pro) 104894588 NM_004870.3(MPDUI):c.2T>C AATAYGGCGGCCGAGGCGGACGG Congenital disorder of glycosylation type 1F (p.Met1Thr) 104894626 NM_000304.3(PMP22):c.82T>C TAGCAAYGGATCGTGGGCAATGG Charcot-Marie-Tooth disease, type IE (p.Trp28Arg) 104894631 NM_018129.3(PNP0):c.784T>C ACCTYAACTCTGGGACCTGCTGG “Pyridoxal 5-phosphate-dependent epilepsy” (p.Ter262Gln) 104894703 NM_032551.4(KISS1R):c.305T>C GCCCTGCYGTACCCGCTGCCCGG, (p.Leu102Pro) TGCYGTACCCGCTGCCCGGCTGG 104894826 NM_000166.5(GJBI):c.407T>C ATGYCATCAGCGTGGTGTTCCGG Dejerine-Sottas disease, X-linked hereditary

motor and sensory neuropathy 104894859 NM_001122606.1(LAIVIP2):c.961T>C CAGCTACYGGGATGCCCCCCTGG, Danon disease (p.Trp321Arg) AGCTACYGGGATGCCCCCCTGGG 104894931 NM_006517.4(SLC16A2):c.1313T>C TGAGCYGGTGGGCCCAATGCAGG Allan-Herndon-Dudley syndrome (p.Leu438Pro) 104894935 NM_000330.3(RS1):c.38T>C TTACTTCYCTTTGGCTATGAAGG Juvenile retinoschisis (p.Leu13Pro) 104895217 NM_001065.3(TNFRSF1A):c.175T>C TGCYGTACCAAGTGCCACAAAGG TNF receptor-associated periodic fever (p.Cys59Arg) syndrome (TRAPS) 143889283 NM_003793.3(CTSF):c.692A>G CTCCAYACTGAGCTGTGCCACGG Ceroid lipofuscinosis, neuronal, 13 (p.Tyr231Cys) 122459147 NM_001159702.2(FHL1):c.310T>C GGGGYGCTTCAAGGCCATTGTGG Myopathy, reducing body, X-linked, (p.Cys104Arg) childhood- onset 74552543 NM_020184.3(CNNM4):c.971T>C AAGCTCCYGGACTTTTTTCTGGG Cone-rod dystrophy amelogenesis imperfecta (p.Leu324Pro) 199476117 m.10158T>C AAAYCCACCCCTTACGAGTGCGG Leigh disease, Leigh syndrome due to mitochondrial complex I deficiency, Mitochondrial complex I deficiency 794727808 NM_020451.2(SEPN1):c.872+2T>C TTCCGGYGAGTGGGCCACACTGG Congenital myopathy with fiber type disproportion, Eichsfeld type congenital muscular dystrophy 140547520 NM_005022.3(PFN1):c.350A>G CACCTYCTTTGCCCATCAGCAGG Amyotrophic lateral sclerosis 18 (p.Glu117Gly) 397514359 NM_000060.3(BTD):c.445T>C TCACCGCYTCAATGACACAGAGG Biotinidase deficiency (p.Phe149Leu) 207460001 m.15197T>C CTAYCCGCCATCCCATACATTGG Exercise intolerance 397514406 NM_000060.3(BTD):c.1214T>C TTCACCCYGGTCCCTGTCTGGGG Biotinidase deficiency (p.Leu405Pro) 397514516 NM_006177.3(NRL):c.287T>C GAGGCCAYGGAGCTGCTGCAGGG Retinitis pigmentosa 27 (p.Met96Thr) 72554312 NM_000531.5(OTC):c.134T>C CTCACTCYAAAAAACTTTACCGG Ornithine carbamoyltransferase deficiency (p.Leu45Pro) 397514569 NM_178012.4(TUBB2B):c.350T>C GGTCCYGGATGTGGTGAGGAAGG Polymicrogyria, asymmetric (p.Leu117Pro) 397514571 NM_000431.3(MVK):c.122T>C CGGCYTCAACCCCACAGCAATGG, Porokeratosis, disseminated superficial actinic (p.Leu41Pro) GGCYTCAACCCCACAGCAATGGG 1 794728390 NM_000238.3(KCNH2):c.2396T>C GCCATCCYGGGTATGGGGTGGGG, Cardiac arrhythmia (p.Leu799Pro) CCATCCYGGGTATGGGGTGGGGG, CATCCYGGGTATGGGGTGGGGGG 397514713 NM_001199107.1(TBC1D24):c.686T>C GGTCTYTGACGTCTTCCTGGTGG Early infantile epileptic encephalopathy 16 (p.Phe229Ser) 397514719 NM_080605.3(B3GALT6):c.193A>G CGCYGGCCACCAGCACTGCCAGG Spondyloepimetaphyseal dysplasia with joint (p.Ser65Gly) laxity 730880608 NM_000256.3(MYBPC3):c.3796T>C GAGYGCCGCCTGGAGGTGCGAGG Cardiomyopathy (p.Cys1266Arg) 397515329 NM_001382.3(DPAGTH:c.503T>C AATCCYGTACTATGTCTACATGG, Congenital disorder of glycosylation type 1J (p.Leu168Pro) ATCCYGTACTATGTCTACATGGG, TCCYGTACTATGTCTACATGGGG 397515465 NM_018127.6(ELAC2):c.460T>C ATAYTTTCTGGTCCATTGAAAGG Combined oxidative phosphorylation deficiency (p.Phe154Leu) 17 397515557 NM_005211.3(CSF1R):c.2483T>C CATCTYTGACTGTGTCTACACGG Hereditary diffuse leukoencephalopathy with (p.Phe828Ser) spheroids 397515599 NM_194248.2(0T0F):c.3413T>C AGGTGCYGTTCTGGGGCCTACGG, Deafness, autosomal recessive 9 (p.Leu1138Pro) GGTGCYGTTCTGGGGCCTACGGG 397515766 NM_000138.4(FBN1):c.2341T>C GGACAAYGTAGAAATACTCCTGG Marfan syndrome (p.Cys781Arg) 565779970 NM_001429.3(EP300):c.3573T>A CTTAYTACAGTTACCAGAACAGG Rubinstein-Taybi syndrome 2 (p.Tyr1191Ter) 786200938 NM_080605.3(B3GALT6):c.1A>G AGCTTCAYGGCGCCCGCGCCGGG, Spondyloepimetaphyseal dysplasia with joint (p.Met1Val) TCAYGGCGCCCGCGCCGGGCCGG laxity 28942087 NM_000229.1(LCAT):c.698T>C ATCTCTCYTGGGGCTCCCTGGGG, Norum disease (p.Leu233Pro) TCTCYTGGGGCTCCCTGGGGTGG 128621203 NM_000061.2(BTK):c.1625T>C TCGGCCYGTCCAGGTGAGTGTGG X-linked agammaglobulinemia with (p.Leu542Pro) growth hormone deficiency 397515412 NM_006383.3(CIB2):c.368T>C CTTCAYCTGCAAGGAGGACCTGG Deafness, autosomal recessive 48 (p.Ile123Thr) 193929364 NM_000352.4(ABCC8):c.404T>C AAGCYGCTAATTGGTAGGTGAGG Permanent neonatal diabetes mellitus (p.Leu135Pro) 730880872 NM_000257.3(MYH7):c.1400T>C TCGAGAYCTTCGATGTGAGTTGG, Cardiomyopathy

CGAGAYCTTCGATGTGAGTTGGG 80356474 NM_002977.3(SCN9A):c.2543T>C AAGATCAYTGGTAACTCAGTAGG, Primary erythromelalgia (p.Ile848Thr) AGATCAYTGGTAACTCAGTAGGG, GATCAYTGGTAACTCAGTAGGGG 80356489 NM_001164277.1(SLC37A4):c.352T>C GGGCYGGCCCCCATGTGGGAAGG Glucose-6-phosphate transport defect (p.Trp118Arg) 80356536 NM_152296.4(ATP1A3):c.2338T>C GCCCYTCCTGCTGTTCATCATGG Dystonia 12 (p.Phe780Leu) 80356596 NM_194248.2(0T0F):c.3032T>C GATGCYGGTGTTCGACAACCTGG Deafness, autosomal recessive 9, Auditory (p.Leu1011Pro) neuropathy, autosomal recessive, 1 80356689 NM_000083.2(CLCN1):c.857T>C AGGAGYGCTATTTAGCATCGAGG Myotonia congenita (p.Val286Ala) 118203884 m.4409T>C AGGYCAGCTAAATAAGCTATCGG Mitochondrial myopathy 587777625 NM_173596.2(SLC39A5):c.911T>C AGAACAYGCTGGGGCTTTTGCGG Myopia 24, autosomal dominant (p.Met304Thr) 587783087 NM_003159.2(CDKL5):c.602T>C ATTCYTGGGGAGCTTAGCGATGG not provided (p.Leu201Pro) 118203951 NM_013319.2(UBIAD1):c.511T>C TCTGGCYCCTTTCTCTACACAGG, Schnyder crystalline corneal dystrophy (p.Ser171Pro) GGCYCCTTTCTCTACACAGGAGG 118204017 NM_000018.3(ACADVL):c.1372T>C TCGCATCYTCCGGATCTTTGAGG, Very long chain acyl-CoA dehydrogenase (p.Phe458Leu) CGCATCYTCCGGATCTTTGAGGG, deficiency GCATCYTCCGGATCTTTGAGGGG 397518466 NM_000833.4(GRIN2A):c.2T>C CTAYGGGCAGAGTGGGCTATTGG Focal epilepsy with speech disorder with or (p.Met1Thr) without mental retardation 118204069 NM_000237.2(LPL):c.337T>C GGACYGGCTGTCACGGGCTCAGG Hyperlipoproteinemia, type I (p.Trp113Arg) 118204080 NM_000237.2(LPL):c.755T>C GTGAYTGCAGAGAGAGGACTTGG Hyperlipoproteinemia, type I (p.Ile252Thr) 118204111 NM_000190.3(HMBS):c.739T>C GCTTCGCYGCATCGCTGAAAGGG Acute intermittent porphyria (p.Cys247Arg) 80357438 NM_007294.3(BRCA1):c.65T>C AAATCTYAGAGTGTCCCATCTGG Familial cancer of breast, Breast-ovarian (p.Leu22Ser) cancer, familial 1, Hereditary cancer-predisposing syndrome 139877390 NM_001040431.2(COA3):c.215A>G CCAYCTGGGGAGGTAGGTTCAGG (p.Tyr72Cys) 793888527 NM_005859.4(PURA):c.563T>C GACCAYTGCGCTGCCCGCGCAGG, not provided, Mental retardation, autosomal (p.Ile188Th) ACCAYTGCGCTGCCCGCGCAGGG, dominant 31 CCAYTGCGCTGCCCGCGCAGGGG 561425038 NM_002878.3(RAD51D):c.1A>G CGCCCAYGTTCCCCGCAGGCCGG Hereditary cancer-predisposing syndrome (p.Met1Val) 121907934 NM_024105.3(ALG12):c.473T>C TCCYGCTGGCCCTCGCGGCCTGG Congenital disorder of glycosylation type 1G (p.Leu158Pro) 80358207 NM_153212.2(GM4):c.409T>C CCTCATCYTCAAGGCCGCCGTGG Erythrokeratodermia variabilis (p.Phe137Leu) 80358228 NM_002353.2(TACSTD2):c.557T>C TCGGCYGCACCCCAAGTTCGTGG Lattice conical dystrophy Type III (p.Leu186Pro) 121908076 NM_138691.2(TMC1):c.1543T>C AGGACCTYGCTGGGAAACAATGG, Deafness, autosomal recessive 7 (p.Cys515Arg) ACCTYGCTGGGAAACAATGGTGG, CCTYGCTGGGAAACAATGGTGGG 121908089 NM_017838.3(NHP2):c.415T>C GGAGGCTYACGATGAGTGCCTGG, Dyskeratosis congenita autosomal recessive 1, (p.Tyr139His) GGCTYACGATGAGTGCCTGGAGG Dyskeratosis congenita, autosomal recessive 2 121908154 NM_001243133.1(NLRP3):c.926T>C GGTGCCTYTGACGAGCACATAGG Familial cold urticaria, Chronic infantile (p.Phe309Ser) neurological, cutaneous and articular syndrome 121908158 NM_001033855.2(DCLRE1C):c.2T>C GGCGCTAYGAGTTCTTTCGAGGG, Histiocytic medullary reticulosis (p.Met1Thr) GCGCTAYGAGTTCTTTCGAGGGG 796052870 NM_018129.3(PNP0):c.2T>C CCCCCAYGACGTGCTGGCTGCGG, not provided (p.Met1Thr) CCCCAYGACGTGCTGGCTGCGGG, CCCAYGACGTGCTGGCTGCGGGG 121908318 NM_020427.2(SLURP1):c.43T>C GCAGCCYGGAGCATGGGCTGTGG Acroerythrokeratoderma (p.Trpl5Arg) 121908352 NM_022124.5(CDH23):c.5663T>C CTCACCTYCAACATCACTGCGGG Deafness, autosomal recessive 12 (p.Phe1888Ser) 121908520 NM_000030.2(AGXT):c.613T>C CCTGTACYCGGGCTCCCAGAAGG Primary hyperoxaluria, type I (p.Ser205Pro) 121908618 NM_004273.4(CHST3):c.920T>C CGTGCYGGCCTCGCGCATGGTGG Spondyloepiphyseal dysplasia with congenital (p.Leu307Pro) joint dislocations 11694 NM_006432.3(NPC2):c.199T>C TATTCAGYCTAAAAGCAGCAAGG Niemann-Pick disease type C2 (p.Ser67Pro) 121908739 NM_000022.2(ADA):c.320T>C CCTGCYGGCCAACTCCAAAGTGG Severe combined immunodeficiency due to (p.Leu107Pro) ADA deficiency 80359022 NM_000059.3(BRCA2):c.7958T>C TGCYTCTTCAACTAAAATACAGG Familial cancer of breast, Breast-ovarian (p.Leu2653Pro) cancer, familial 2 121908902 NM_003880.3(WISP3):c.232T>C AAAATCYGTGCCAAGCAACCAGG, Progressive pseudorheumatoid dysplasia (p.Cys78Arg) AAATCYGTGCCAAGCAACCAGGG, AATCYGTGCCAAGCAACCAGGGG 121908947 NM_006892.3(DNMT3B):c.808T>C CAAGTTCYCCGAGGTGAGTCCGG, Centromeric instability of chromosomes 1,9 and (p.Ser270Pro) AAGTTCYCCGAGGTGAGTCCGGG, 16 and immunodeficiency AGTTCYCCGAGGTGAGTCCGGGG 121909028 NM_000492.3(CFTR):c.3857T>C AGCCTYTGGAGTGATACCACAGG Cystic fibrosis (p.Phe1286Ser) 121909135 NM_000085.4(CLCNKB):c.1294T>C CTTTGTCYATGGTGAGTCTGGGG Bartter syndrome type 3 (p.Tyr432His) 121909143 NM_001300.5(KLF6):c.506T>C GGAGCYGCCCTCGCCAGGGAAGG (p.Leu169Pro) 121909182 NM_001089.2(ABCA3):c.302T>C GCACYTGTGATCAACATGCGAGG Surfactant metabolism dysfunction, pulmonary, (p.Leu101Pro) 3 121909200 NM_000503.5(EYA1):c.1459T>C CACTCYCGCTCATTCACTCCCGG Melnick-Fraser syndrome (p.Ser487Pro) 121909247 NM_004970.2(IGFALS):c.1618T>C GGACYGTGGCTGCCCTCTCAAGG Acid-labile subunit deficiency (p.Cys540Arg) 121909253 NM_005570.3(LMAN1):c.2T>C AGAYGGCGGGATCCAGGCAAAGG Combined deficiency of factor V and factor (p.Met1Thr) VIII, 1 121909385 NM_000339.2(SLC12A3):c.1868T>C CAACCYGGCCCTCAGCTACTCGG Familial hypokalemia-hypomagnesemia (p.Leu623Pro) 121909497 NM_002427.3(MMP13):c.224T>C TTCTYCGGCTTAGAGGTGACTGG Spondyloepimetaphyseal dysplasia, Missouri (p.Phe75Ser) type 121909508 NM_000751.2(CHRND):c.188T>C AACCYCATCTCCCTGGTGAGAGG MYASTHENIC SYNDROME, (p.Leu63Pro) CONGENITAL, 3B, FAST- CHANNEL 121909519 NM_001100.3(ACTA1):c.287T>C CGAGCYTCGCGTGGCTCCCGAGG Nemaline myopathy 3 (p.Leu96Pro) 121909572 NM_000488.3(SERPINC1):c.667T>C TGGGTGYCCAATAAGACCGAAGG Antithrombin III deficiency (p.Ser223Pro) 121909677 NM_000821.6(GGCX):c.896T>C TATGTYCTCCTACGTCATGCTGG Pseudoxanthoma elasticum-like disorder with (p.Phe299Ser) multiple coagulation factor deficiency 121909727 NM_001018077.1(NR3C1):c.2209T>C CTATTGCYTCCAAACATTTTTGG Glucocorticoid resistance, generalized (p.Phe737Leu) 139573311 NM_000492.3(CFTR):c.1400T>C TTCACYTCTAATGGTGATTATGG, Cystic fibrosis (p.Leu467Pro) TCACYTCTAATGGTGATTATGGG 121912441 NM_000454.4(SOD1):c.341T>C CATCAYTGGCCGCACACTGGTGG Amyotrophic lateral sclerosis type 1 (p.Ile114Thr) 121912446 NM_000454.4(SOD1):c.434T>C CGTTYGGCTTGTGGTGTAATTGG, Amyotrophic lateral sclerosis type 1 (p.Leu145Ser) GTTYGGCTTGTGGTGTAATTGGG 121912463 NM_000213.3(ITGB4):c.1684T>C GGCCAGYGTGTGTGTGAGCCTGG Epidermolysis bullosa with pyloric atresia (p.Cys562Arg) 121912492 NM_002292.3(LAIVIB2):c.961T>C CCTCAACYGCGAGCAGTGTCAGG Nephrotic syndrome, type 5, with or (p.Cys321Arg) without ocular abnormalities 397516659 NM_001399.4(EDA):c.2T>C GGCCAYGGGCTACCCGGAGGTGG Hypohidrotic X-linked ectodermal dysplasia (p.Met1Thr) 111033589 NM_021044.2(DHH):c.485T>C GTTGCYGGCGCGCCTCGCAGTGG 46, XY gonadal dysgenesis, complete, dhh- (p.Leu162Pro) related 111033622 NM_000206.2(IL2RG):c.343T>C TGGCYGTCAGTTGCAAAAAAAGG X-linked severe combined immunodeficiency (p.Cys115Arg) 121912613 NM_001041.3(SI):c.1859T>C ATGCYGGAGTTCAGTTTGTTTGG Sucrase-isomaltase deficiency (p.Leu620Pro) 121912619 NM_016180.4(SLC45A2):c.1082T>C GAGTTTCYCATCTACGAAAGAGG Oculocutaneous albinism type 4 (p.Leu361Pro) 61750581 NM_000552.3(VWF):c.4837T>C CTGCCYCTGATGAGATCAAGAGG von Willebrand disease, type 2a (p.Ser1613Pro) 121912653 NM_000546.5(TP53):c.755T>C CATCCYCACCATCATCACACTGG Li-Fraumeni syndrome 1 (p.Leu252Pro) 111033683 NM_000155.3(GALT):c.386T>C AGGTCAYGTGCTTCCACCCCTGG Deficiency of UDPglucose-hexose-1-phosphate (p.Met129Thr) uridylyltransferase 111033752 NM_000155.3(GALT):c.677T>C CAGGAGCYACTCAGGAAGGTGGG Deficiency of UDPglucose-hexose-1-phosphate (p.Leu226Pro) uridylyltransferase 121912729 NM_000039.1(AP0A1):c.593T>C GCGCTYGGCCGCGCGCCTTGAGG Familial visceral amyloidosis, Ostertag type (p.Leu198Ser) 769452 NM_000041.3(APOE):c.137T>C AACYGGCACTGGGTCGCTTTTGG (p.Leu46Pro) 121912762 NM_016124.4(RHD):c.329T>C ACACYGTTCAGGTATTGGGATGG (p.Leu110Pro) 111033824 NM_000155.3(GALT):c.1138T>C CGCCYGACCACGCCGACCACAGG, Deficiency of UDPglucose-hexose-1-phosphate (p.Ter380Arg) GCCYGACCACGCCGACCACAGGG uridylyltransferase 111033832 NM_000155.3(GALT):c.980T>C TCCYGCGCTCTGCCACTGTCCGG Deficiency of UDPglucose-hexose-1-phosphate (p.Leu327Pro) uridylyltransferase 730881974 NM_000455.4(STK11):c.545T>C GGGAACCYGCTGCTCACCACCGG, Hereditary cancer-predisposing syndrome (p.Leu182Pro) AACCYGCTGCTCACCACCGGTGG 1064644 NM_000157.3(GBA):c.703T>C GGGYCACTCAAGGGACAGCCCGG Gaucher disease (p.Ser235Pro) 796052090 NM_138413.3(HOGA1):c.533T>C GGACCYGCCTGTGGATGCAGTGG Primary hyperoxaluria, type III (p.Leu178Pro) 121913141 NM_000208.2(INSR):c.779T>C CTACCYGGACGGCAGGTGTGTGG Leprechaunism syndrome (p.Leu260Pro) 121913272 NM_006218.2(PIK3CA):c.1258T>C GGAACACYGTCCATTGGCATGGG, Congenital lipomatous overgrowth, vascular (p.Cys420Arg) GAACACYGTCCATTGGCATGGGG malformations, and epidermal nevi, Neoplasm of ovary, PIK3CA Related Overgrowth Spectrum 61751310 NM_000552.3(VWF):c.8317T>C GCTCCYGCTGCTCTCCGACACGG von Willebrand disease, type 2a (p.Cys2773Arg) 312262799 NM_024408.3(NOTCH2):c.1438T>C TTCACAYGTCTGTGCATGCCAGG Alagille syndrome 2 (p.Cys480Arg) 121913570 NM_000426.3(LAIVIA2):c.7691T>C ATCATTCYTTTGGGAAGTGGAGG, Merosin deficient congenital (p.Leu2564Pro) TCATTCYTTTGGGAAGTGGAGGG muscular dystrophy 121913640 NM_000257.3(MYH7):c.1046T>C AACTCCAYGTATAAGCTGACAGG Familial hypertrophic cardiomyopathy (p.Met349Thr) 1, Cardiomyopathy 121913642 NM_000257.3(MYH7):c.1594T>C CATCATGYCCATCCTGGAAGAGG Dilated cardiomyopathy 1S (p.Ser532Pro) 119463996 NM_001079802.1(FKTN):c.527T>C GTAGTCTYTCATGAGAGGAGTGG Limb-girdle muscular (p.Phe176Ser) dystrophy- 587776456 NM_002049.3(GATA1):c.1240T>C GCTCAYGAGGGCACAGAGCATGG GATA-1-related thrombocytopenia (p.Ter414Arg) with dyserythropoiesis 63750654 NM_000184.2(HBG2):c.-228T>C ATGCAAAYATCTGTCTGAAACGG Fetal hemoglobin quantitative trait locus 1 587776519 NM_001999.3(FBN2):c.3725-15A>G AGCAYTGCAACCACATTGTCAGG Congenital contractural arachnodactyly 78365220 NM_000402.4(G6PD):c.473T>C TGCCCYCCACCTGGGGTCACAGG Anemia, nonspherocytic hemolytic, due to (p.Leu158Pro) G6PD deficiency 63750741 NM_000179.2(MSH6):c.1346T>C CTGGGGCYGGTATTCATGAAAGG Hereditary Nonpolyposis Colorectal Neoplasms (p.Leu449Pro) 587776914 NM_017565.3(FAIVI20A):c.590-2A>G GTAATCYGCAAAGGAGGAGAAGG, Enamel-renal syndrome TAATCYGCAAAGGAGGAGAAGG 5030809 NM_000551.3(VHL):c.292T>C CCCYACCCAACGCTGCCGCCTGG Von Hippel-Lindau syndrome, (p.Tyr98His) Hereditary cancer-predisposing syndrome 199476132 m.5728T>C CAATCYACTTCTCCCGCCGCCGG, Cytochrome-c oxidase deficiency, Mitochondrial AATCYACTTCTCCCGCCGCCGGG complex I deficiency 62637012 NM_014336.4(AIPL1):c.715T>C CTGCCAGYGCCTGCTGAAGAAGG, Leber congenital amaurosis 4 (p.Cys239Arg) CCAGYGCCTGCTGAAGAAGGAGG 199476199 NM_207352.3(CYP4V2):c.1021T>C AAACTGGYCCTTATACCTGTTGG, Bietti crystalline comeoretinal dystrophy (p.Ser341Pro) AACTGGYCCTTATACCTGTTGGG 587777183 NM_006702.4(PNPLA6):c.3053T>C CCTYTAACCGCAGCATCCATCGG Boucher Neuhauser syndrome (p.Phe1018Ser) 199476389 NM_000487.5(ARSA):c.899T>C GGTCTCTYGCGGTGTGGAAAGGG Metachromatic leukodystrophy (p.Leu300Ser) 199476398 NM_016599.4(MYOZ2):c.142T>C TTAYCCCATCTCAGTAACCGTGG Familial hypertrophic cardiomyopathy 16 (p.Ser48Pro) 119456967 NM_001037633.1(SIL1):c.1370T>C TTGCYGAAGGAGCTGAGATGAGG Marinesco-Sj\xc3\xb6gren syndrome (p.Leu457Pro) 730882253 NM_006888.4(CALM1):c.268T>C GGCAYTCCGAGTCTTTGACAAGG Long QT syndrome 14 (p.Phe90Leu) 587777283 NM_012338.3(TSPAN12):c.413A>G TAATCCAYAATTTGTCATCCTGG Exudative vitreoretinopathy 5 (p.Tyr138Cys) 587777306 NM_015884.3(MBTPS2):c.1391T>C GCTYTGCTTTGGATGGACAATGG Palmoplantar keratoderma, mutilating, with (p.Phe464Ser) periorificial keratotic plaques, X-linked 56378716 NM_000250.1(MPO):c.752T>C TCACTCAYGTTCATGCAATGGGG Myeloperoxidase deficiency (p.Met251Thr) 587777390 NM_005026.3(PIK3CD):c.1246T>C GCAGGACYGCCCCATTGCCTGGG Activated PI3K-delta syndrome (p.Cys416Arg) 587777480 NM_003108.3(SOXI 1 ):c.I78T>C TATGGYCCAAGATCGAACGCAGG Mental retardation, autosomal dominant 27 (p.Ser60Pro) 587777663 NM_001288767.1(ARMC5):c.1379T>C GCCCGACYGCGGGATGCTGGTGG Acth-independent macronodular adrenal (p.Leu460Pro) hyperplasia 2 61753033 NM_000350.2(ABCA4):c.5819T>C AAGGCYACATGAACTAACCAAGG Stargardt disease, Stargardt disease 1, Cone-rod dystrophy 3 (p.Leu1940Pro) 200488568 NM_002972.3(SBF1):c.4768A>G CAGGCGYCCTCTTGCTCAGCCGG Charcot-Marie-Tooth disease, type 4B3 (p.Thr1590Ala) 132630274 NM_000377.2(WAS):c.809T>C CGGAGTCYGTTCTCCAGGGCAGG Severe congenital neutropenia X-linked (p.Leu270Pro) 132630308 NM_001399.4(EDA):c.181T>C CTGCYACCTAGAGTTGCGCTCGG Hypohidrotic X-linked ectodermal dysplasia (p.Tyr61His) 60934003 NM_170707.3(LMNA):c.1589T>C ACGGCTCYCATCAACTCCACTGG, Benign scapuloperoneal muscular dystrophy (p.Leu530Pro) CGGCTCYCATCAACTCCACTGGG, with cardiomyopathy GGCTCYCATCAACTCCACTGGGG 180177160 NM_000030.2(AGXT):c.1076T>C GGTGCYGCGGATCGGCCTGCTGG, Primary hyperoxaluria, type I (p.Leu359Pro) GTGCYGCGGATCGGCCTGCTGGG 180177222 NM_000030.2(AGXT):c.449T>C GTGCYGCTGTTCTTAACCCACGG, Primary hyperoxaluria, type I (p.Leu150Pro) TGCYGCTGTTCTTAACCCACGGG 180177254 NM_000030.2(AGXT):c.661T>C GCTCATCYCCTTCAGTGACAAGG Primary hyperoxaluria, type I (p.Ser221Pro) 180177264 NM_000030.2(AGXT):c.757T>C GGGGCYGTGACGACCAGCCCAGG Primary hyperoxaluria, type I (p.Cys253Arg) 180177293 NM_000030.2(AGXT):c.893T>C GTATCYGCATGGGCGCCTGCAGG Primary hyperoxaluria, type I (p.Leu298Pro) 376785840 NM_001282227.1(CECR1):c.1232A>G GAAATCAYAGGACAAGCCTTTGG Polyarteritis nodosa (p.Tyr411Cys) 587779393 NM_000257.3(MYH7):c.4937T>C GAGCCYCCAGAGCTTGTTGAAGG Myopathy, distal, 1 (p.Leu1646Pro) 587779410 NM_012434.4(SLC17A5):c.500T>C ATTGTACYCAGAGCACTAGAAGG Sialic acid storage disease, severe infantile (p.Leu167Pro) type 587779513 NM_000090.3(COL3A1):c.2337+2T>C AGGYAACCCTTAATACTACCTGG Ehlers-Danlos syndrome, type 4 (p.Gly762_Lys779del) 777539013 NM_020376.3(PNPLA2):c.757+2T>C GAACGGYGCGCGGACCCGGGCGG, Neutral lipid storage disease with myopathy AACGGYGCGCGGACCCGGGCGGG 34557412 NM_012452.2(TNFRSF13B):c.310T>C ACTTCYGTGAGAACAAGCTCAGG Immunoglobulin A deficiency 2, (p.Cys104Arg) Common variable 796052970 NM_001165963.1(SCN1A):c.1094T>C CAAGCTYTGATACCTTCAGTTGG, not provided (p.Phe365Ser) AAGCTYTGATACCTTCAGTTGGG 724159989 NC_012920.1:m.7505T>C CCTCCAYGACTTTTTCAAAAAGG Deafness, nonsyndromic sensorineural, mitochondrial 796053222 NM_014191.3(SCN8A):c.4889T>C CGTCYGATCAAAGGCGCCAAAGG, not provided (p.Leu1630Pro) GTCYGATCAAAGGCGCCAAAGGG 118192127 NM_000540.2(RYR1):c.10817T>C TACTACCYGGACCAGGTGGGTGG, Central core disease (p.Leu3606Pro) ACTACCYGGACCAGGTGGGTGGG, CTACCYGGACCAGGTGGGTGGGG 118192170 NM_000540.2(RYR1):c.14693T>C AGGCAYTGGGGACGAGATCGAGG Malignant hyperthermia susceptibility type 1, (p.Ile4898Thr) Central core disease 121917703 NM_005247.2(FGF3):c.466T>C GTACGTGYCTGTGAACGGCAAGG, Deafness with labyrinthine aplasia microtia and (p.Ser156Pro) TACGTGYCTGTGAACGGCAAGGG microdontia (LAIVIM) 690016549 NM_005211.3(CSF1R):c.2450T>C CCGCCYGCCTGTGAAGTGGATGG Hereditary diffuse leukoencephalopathy h (p.Leu817Pro) spheroids 690016552 NM_005211.3(CSF1R):c.2566T>C GAATCCCYACCCTGGCATCCTGG Hereditary diffuse leukoencephalopathy with (p.Tyr856His) spheroids 121917738 NM_001098668.2(SFTPA2):c.593T>C GGAGACTYCCGCTACTCAGATGG, Idiopathic fibrosing alveolitis, chronic form (p.Phe198Ser) GAGACTYCCGCTACTCAGATGGG 690016559 NM_005211.3(CSF1R):c.1957T>C AGCCYGTACCCATGGAGGTAAGG, Hereditary diffuse leukoencephalopathy with (p.Cys653Arg) GCCYGTACCCATGGAGGTAAGGG spheroids 690016560 NM_005211.3(CSFIR):c.2717T>C GCAGAYCTGCTCCTTCCTTCAGG Hereditary diffuse leukoencephalopathy with (p.Ile906Thr) spheroids 121917769 NM_003361.3(UMOD):c.376T>C GGCCACAYGTGTCAATGTGGTGG, Familial juvenile gout (p.Cys126Arg) GCCACAYGTGTCAATGTGGTGGG 121917773 NM_003361.3(UMOD):c.943T>C ATGGCACYGCCAGTGCAAACAGG Glomerulocystic kidney disease (p.Cys315Arg) with hyperuricemia and isosthenuria 121917818 NM_007255.2(B4GALT7):c.617T>C TGCYCTCCAAGCAGCACTACCGG Ehlers-Danlos syndrome progeroid type (p.Leu206Pro) 121917824 NM_021615.4(CHST6):c.827T>C GGACCYGGCGCGGGAGCCGCTGG Macular corneal dystrophy Type I (p.Leu276Pro) 121917848 NM_000452.2(SLC10A2):c.728T>C TTTCYTCTGGCTAGAATTGCTGG Bile acid malabsorption, primary (p.Leu243Pro) 121918006 NM_000478.4(ALPL):c.1306T>C TGGACYATGGTGAGACCTCCAGG Infantile hypophosphatasia (p.Tyr436His) 121918010 NM_000478.4(ALPL):c.979T>C CAAAGGCYTCTTCTTGCTGGTGG, Infantile hypophosphatasia (p.Phe327Leu) GGCYTCTTCTTGCTGGTGGAAGG 121918088 NM_000371.3(TTR):c.400T>C CCCCYACTCCTATTCCACCACGG (p.Tyr134His) 121918110 NM_001042465.1(PSAP):c.1055T>C GAAGCYGCCGAAGTCCCTGTCGG Gaucher disease, atypical, due to saposin C (p.Leu352Pro) deficiency 121918137 NM_003730.4(RNASET2):c.550T>C CCAGYGCCTTCCACCAAGCCAGG Leukoencephalopathy, cystic, (p.Cys184Arg) without megalencephaly 121918191 NM_001127628.1(FBP1):c.581T>C GGAGTYCATTTTGGTGGACAAGG Fructose-biphosphatase deficiency (p.Phe194Ser) 121918306 NM_006946.2(SPTBN2):c.758T>C ACCAAGCYGCTGGATCCCGAAGG, Spinocerebellar ataxia 5 (p.Leu253Pro) AAGCYGCTGGATCCCGAAGGTGG, AGCYGCTGGATCCCGAAGGTGGG 121918505 NM_000141.4(FGFR2):c.799T>C AATGCCYCCACAGTGGTCGGAGG Pfeiffer syndrome, Neoplasm of stomach (p.Ser267Pro) 121918643 NM_003126.2(SPTAI):c.620T>C GTGGAGCYGGTAGCTAAAGAAGG, Hereditary pyropoikilocytosis, Elliptocytosis 2 (p.Leu207Pro) TGGAGCYGGTAGCTAAAGAAGGG 121918646 NM_001024858.2(SPTB):c.604T>C CTCCAGCYGGAAGGATGGCTTGG Spherocytosis type 2 (p.Trp202Arg) 121918648 NM_001024858.2(SPTB):c.6055T>C ATGCCYCTGTGGCTGAGGCGTGG (p.Ser2019Pro) 727504166 NM_000543.4(SMPD1):c.475T>C TGAGGCCYGTGGCCTGCTCCTGG, Niemann-Pick disease, type A, Niemann-Pick (p.Cys159Arg) GAGGCCYGTGGCCTGCTCCTGGG disease, type B 193922915 NM_000434.3(NEU1):c.1088T>C CAGCYATGGCCAGGCCCCAGTGG Sialidosis, type II (p.Leu363Pro) 727504419 NM_000501.3(ELN):c.889+2T>C CAGGYAACATCTGTCCCAGCAGG, Supravalvar aortic stenosis AGGYAACATCTGTCCCAGCAGGG 376395543 NM_000256.3(MYBPC3):c.26- GAGACYGAAGGGCCAGGTGGAGG Primary familial hypertrophic 2A>G cardiomyopathy, Familial hypertrophic cardiomyopathy 4, Cardiomyopathy 1169305 NM_000545.6(RNIF1A):c.1720G>A GATGCYGGCAGGGTCCTGGCTGG, Maturity-onset diabetes of the young, type 3 (p.Gly574Ser) ATGCYGGCAGGGTCCTGGCTGGG, TGCYGGCAGGGTCCTGGCTGGGG 730880130 NM_000527.4(LDLR):c.1468T>C CTACYGGACCGACTCTGTCCTGG, Familial hypercholesterolemia (p.Trp490Arg) TACYGGACCGACTCTGTCCTGGG 281860286 NM_018713.2(SLC30A10):c.500T>C GGCGCTTYCGGGGGGCCTCAGGG Hypermanganesemia with dystonia, (p.Phe167Ser) polycythemia and cirrhosis 730880306 NM_145693.2(LPIN1):c.441+2T>C AAGGYACCGCGGGCCTCGCGCGG, Myoglobinuria, acute recurrent, autosomal AGGYACCGCGGGCCTCGCGCGGG recessive 74315452 NM_000454.4(SOD1):c.338T>C TTGCAYCATTGGCCGCACACTGG Amyotrophic lateral sclerosis type 1 (p.Ile113Thr) 730880455 NM_000169.2(GLA):c.4IT>C CGCGCYTGCGCTTCGCTTCCTGG not provided (p.Leul4Pro) 267606656 NM_054027.4(ANKH):c.1015T>C AGCTCYGTTTCGTGATGTTTTGG Craniometaphyseal dysplasia, autosomal (p.Cys339Arg) dominant 267606687 NM_033409.3(SLC52A3):c.1238T>C AGTTACGYCAAGGTGATGCTGGG Brown-Vialetto-Van laere syndrome (p.Val413Ala) 267606721 NM_001928.2(CFD):c.640T>C GGTGYGCGGGGGCGTGCTCGAGG, Complement factor d deficiency (p.Cys214Arg) GTGYGCGGGGGCGTGCTCGAGGG 267606747 NM_001849.3(COL6A2):c.2329T>C CGCCYGCGACAAGCCACAGCAGG Ullrich congenital muscular dystrophy (p.Cys777Arg) 431905515 NM_001044.4(SLC6A3):c.671T>C CTGCACCYCCACCAGAGCCATGG Infantile Parkinsonism-dystonia (p.Leu224Pro) 267606857 NM_000180.3(GUCY2D):c.2846T>C AGAGAYCGCCAACATGTCACTGG Cone-rod dystrophy 6 (p.Ile949Thr) 267606880 NM_022489.3(INF2):c.125T>C GCTGCYCCAGATGCCCTCTGTGG Focal segmental glomerulosclerosis 5 (p.Leu42Pro) 515726191 NM_015713.4(RRM2B):c.581A>G AACTCCTYCTACAGCAGCAAAGG RRM2B-related mitochondrial disease (p.Glu194Gly) 267606917 NM_004646.3(NPHS1):c.793T>C GCTGCCGYGCGTGGCCCGAGGGG, Finnish congenital nephrotic syndrome (p.Cys265Arg) CTGCCGYGCGTGGCCCGAGGGGG 267607104 NM_001199107.1(TBC1D24):c.751T>C CAAGTTCYTCCACAAGGTGAGGG, Myoclonic epilepsy, familial infantile (p.Phe251Leu) TTCYTCCACAAGGTGAGGGCCGG 267607182 NM_144631.5(ZNF513):c.1015T>C TGGGCGCYGCATGCGAGGAGAGG, Retinitis pigmentosa 58 (p.Cys339Arg) CGCYGCATGCGAGGAGAGGCTGG 267607211 NM_000229.1(LCAT):c.508T>C TATGACYGGCGGCTGGAGCCCGG Norum disease (p.Trp170Arg) 267607215 NM_016269.4(LEF1):c.181T>C GAACGAGYCTGAAATCATCCCGG Sebaceous tumors, somatic (p.Ser61Pro} 587783580 NM_178151.2(DCX):c.683T>C AAAAAACYCTACACTCTGGATGG Heterotopia (p.Leu228Pro) 587783644 NM_004004.5(GM2):c.107T>C GATCCYCGTTGTGGCTGCAAAGG Hearing impairment (p.Leu36Pro) 587783653 NM_005682.6(ADGRG1):c.1460T>C CCCTGCYCACCTGCCTTTCCTGG Polymicrogyria, bilateral frontoparietal (p.Leu487Pro) 587783863 NM_000252.2(MTM1):c.958T>C GGAAYCTTTAAAAAAAGTGAAGG Severe X-linked myotubular myopathy (p.Ser320Pro) 267607751 NM_000249.3(MLH1):c.453+2T>C ATCACGGYAAGAATGGTACATGG, Hereditary Nonpolyposis Colorectal Neoplasms TCACGGYAAGAATGGTACATGGG 119103227 NM_000411.6(HLCS):c.710T>C CTATCYTTCTCAGGGAGGGAAGG Holocarboxylase synthetase deficiency (p.Leu237Pro) 119103237 NM_005787.5(ALG3):c.211T>C GATTGACYGGAAGGCCTACATGG Congenital disorder of glycosylation type 1D (p.Trp7lArg) 398122806 NM_003172.3(SURF1):c.679T>C CCACYGGCATTATCGAGACCTGG Congenital myasthenic syndrome, (p.Trp227Arg) acetazolamide-responsive 80338747 NM_004525.2(LRP2):c.7564T>C GTACCTGYACTGGGCTGACTGGG Donnai Barrow syndrome (p.Tyr2522His) 398122838 NM_001271723.1(FBX038):c.616T>C TTCCTYGTATCCCAATGCTAAGG Distal hereditary motor neuronopathy 2D (p.Cys206Arg) 398122989 NM_014495.3(ANGPTL3):c.883T>C ACAAAACYTCAATGAAACGTGGG Hypobetalipoproteinemia, familial, 2 (p.Phe295Leu) 80338945 NM_004004.5(GM2):c.269T>C GCTCCYAGTGGCCATGCACGTGG Deafness, autosomal recessive 1A, Hearing (p.Leu90Pro) impairment 80338956 NM_000334.4(SCN4A):c.2078T>C AAGATCAYTGGCAATTCAGTGGG, Hyperkalemic Periodic Paralysis Type 1, (p.Ile693Thr) AGATCAYTGGCAATTCAGTGGGG, Paramyotonia congenita of von Eulenburg GATCAYTGGCAATTCAGTGGGGG 267608131 NM_000179.2(MSH6):c.4001+2T>C CGGYAACTAACTAACTATAATGG Hereditary Nonpolyposis Colorectal Neoplasms 587784573 NM_004963.3(GUCY2C):c.2782T>C TCCCYGTGCTGCTGGAGTTGTGG, Meconium ileus (p.Cys928Arg) CCCYGTGCTGCTGGAGTTGTGGG 267608511 NM_003159.2(CDKL5):c.659T>C CCAACYTTTTACTATTCAGAAGG Early infantile epileptic encephalopathy 2 (p.Leu220Pro) 373842615 NM_000118.3(ENG):c.1273-2A>G CCGCCYGCGGGGATAAAGCCAGG, Haemorrhagic telangiectasia 1 CGCCYGCGGGGATAAAGCCAGGG 185492581 NM_000335.4(SCN5A):c.376A>G GAATCTYCACAGCCGCTCTCCGG Brugada syndrome (p.Lys126Glu) 200533370 NM_133499.2(SYN1):c.1699A>G GATGYCTGACGGGTAGCCTGTGG, Epilepsy, X-linked, with variable learning (p.Thr567Ala) ATGYCTGACGGGTAGCCTGTGGG disabilities and behavior disorders, not specified 118203981 NM_148960.2(CLDN19):c.269T>C GCTCCYGGGCTTCGTGGCCATGG Hypomagnesemia 5, renal, with ocular (p.Leu90Pro) involvement 137853892 NM_001235.3(SERPINH1):c.233T>C GTCGCYAGGGCTCGTGTCGCTGG, Osteogenesis imperfecta type 10 (p.Leu78Pro) TCGCYAGGGCTCGTGTCGCTGGG 118204024 NM_000263.3(NAGLU):c.142T>C GGCCGACYTCTCCGTGTCGGTGG Mucopolysaccharidosis,MPS-III-B 690016563 NM_005211.3(CSF1R):c.1745T>C CAACCYGCAGTTTGGTGAGATGG Hereditary diffuse leukoencephalopathy with (p.Leu582Pro) spheroids 58380626 NM_000526.4(KRT14):c.1243T>C CGCCACCYACCGCCGCCTGCTGG, Epidermolysis bullosa herpetiformis, (p.Tyr415His) CACCYACCGCCGCCTGCTGGAGG, Dowling- Meara ACCYACCGCCGCCTGCTGGAGGG 113994151 NM_207346.2(TSEN54):c.277T>C TTGAAGYCTCCCGCGGTGAGCGG, Pontocerebellar hypoplasia type 4 (p.Ser93Pro) AAGYCTCCCGCGGTGAGCGGCGG 113994206 NM_004937.2(CTNS):c.473T>C TGGTCYGAGCTTCGACTTCGTGG Cystinosis (p.Leu158Pro) 62516109 NM_000277.1(PAH):c.638T>C CCACTTCYTGAAAAGTACTGTGG Phenylketonuria (p.Leu213Pro) 370011798 NM_001302946.1(TRNT1):c.668T>C GCAAYTGCAGAAAATGCAAAAGG Sideroblastic anemia with B-cell (p.Ile223Thr) immunodeficiency, periodic fevers, and developmental delay 62517167 NM_000277.1(PAH):c.293T>C AAGATCTYGAGGCATGACATTGG Mild non-PKU hyperphenylalanem a (p.Leu98Ser) 12021720 NM_001918.3(DBT):c.1150G>A GACYCACAGAGCCCAATTTCTGG Intermediate maple syrup urine disease type 2 (p.Gly384Ser) 104886289 NM_000495.4(COL4A5):c.4756T>C TCCCCATYGTCCTCAGGGATGGG Alport syndrome, X-linked recessive (p.Cys1586Arg) 370471013 NC_012920.1:m.5559A>G CAACYTACTGAGGGCTTTGAAGG Leigh disease 121434215 NM_000487.5(ARSA):c.410T>C GCCTTCCYGCCCCCCCATCAGGG Metachromatic leukodystrophy, adult type (p.Leu137Pro) 386134128 NM_000096.3(CP):c.1123T>C ACACTACYACATTGCCGCTGAGG Deficiency of ferroxidase (p.Tyr375His) 121434275 NM_001127328.2(ACADM):c.1136T>C GTGCAGAYACTTGGAGGCAATGG Medium-chain acyl-coenzyme (p.Ile379Thr) A dehydrogenase deficiency 121434276 NM_001127328.2(ACADM):c.1136T>C CAGCGAYGTTCAGATACTAGAGG Medium-chain acyl-coenzyme (p.Cys248Arg) A dehydrogenase deficiency 121434284 NM_002225.3(IVD):c.134T>C ATGGGCYAAGCGAGGAGCAGAGG ISOVALERIC ACIDEMIA, TYPE I (p.Leu45Pro) 121434334 NM_005908.3(MANBA):c.1513T>C ATTACGYCCAGTCCTACAAATGG, Beta-D-mannosidosis (p.Ser505Pro) TTACGYCCAGTCCTACAAATGGG, TACGYCCAGTCCTACAAATGGGG 121434366 NM_000159.3(GCDH):c.883T>C CGCCCGGYACGGCATCGCGTGGG, Glutaric aciduria, type 1 (p.Tyr295His) GCCCGGYACGGCATCGCGTGGGG 60715293 NM_000424.3(KRT5):c.541T>C GTTTGCCYCCTTCATCGACAAGG Epidermolysis bullosa herpetiformis, (p.Ser181Pro) Dowling-Meara 121434409 NM_001003722.1(GLE1):c.2051T>C AAGGACAYTCCTGTCCCCAAGGG Lethal arthrogryposis with anterior horn cell (p.Ile684Thr) disease 121434434 NM_001287.5(CLCN7):c.2297T>C GGGCCYGCGGCACCTGGTGGTGG Osteopetrosis autosomal recessive 4 (p.Leu766Pro) 121434455 NM_000466.2(PEX1):c.1991T>C GATGACCYTGACCTCATTGCTGG Zellweger syndrome (p.Leu664Pro) 199422317 NM_001099274.1(TINF2):c.862T>C CTGYTTCCCTTTAGGAATCTCGG Aplastic anemia (p.Phe288Leu) 104895221 NM_001065.3(TNFRSF1A):c.349T>C CTCTTCTYGCACAGTGGACCGGG TNF receptor-associated periodic fever (p.Cys117Arg) syndrome (TRAPS) 137854459 NM_000138.4(FBN1):c.4987T>C GGGACAYGTTACAACACCGTTGG Marfan syndrome (p.Cys1663Arg) 387907075 NM_024027.4(COLEC11):c.505T>C CAGCTGYCCTGCCAGGGCCGCGG, Carnevale syndrome (p.Ser169Pro) AGCTGYCCTGCCAGGGCCGCGGG, GCTGYCCTGCCAGGGCCGCGGGG, CTGYCCTGCCAGGGCCGCGGGGG 1048095 NM_000352.4(ABCC8):c.674T>C TGCYGTCCAAAGGCACCTACTGG Permanent neonatal diabetes mellitus (p.Leu225Pro) 796065347 NM_019074.3(DLL4):c.1168T>C GAAYGTCCCCCCAACTTCACCGG Adams-Oliver syndrome, ADAMS- (p.Cys390Arg) OLIVER SYNDROME 6 137852347 NM_000402.4(G6PD):c.1054T>C AGGGYACCTGGACGACCCCACGG Anemia, nonspherocytic hemolytic, due to (p.Tyr352His) G6PD deficiency 74315327 NM_213653.3(HFE2):c.302T>C GGACCYCGCCTTCCATTCGGCGG Hemochromatosis type 2A (p.Leu101Pro) 137852579 NM_000044.3(AR):c.2033T>C GTCCYGGAAGCCATTGAGCCAGG (p.Leu678Pro) 137852636 NM_001166107.1(IIMGCS2):c.520T>C CCCTCYTCAATGCTGCCAACTGG mitochondria 3-hydroxy-3-methylglutaryl- (p.Phe174Leu) CoA synthase deficiency 137852661 NM_033163.3(FGF8):c.118T>C TTCCCTGYTCCGGGCTGGCCGGG Kallmann syndrome 6 (p.Phe40Leu) 121912967 NM_005215.3(DCC):c.503T>C AGCCCAYGCCAACAATCCACTGG (p.Met168Thr) 137852806 NM_001039523.2(CHRNA1):c.901T>C TGTGYTCCTTCTGGTCATCGTGG Myasthenic syndrome, congenital, fast-channel (p.Phe301Leu) 137852850 NM_182760.3(SUMF1):c.463T>C GGCGACYCCTTTGTCTTTGAAGG Multiple sulfatase deficiency (p.Ser155Pro) 137852886 NM_000158.3(GBE1):c.671T>C AATGTACYACCAAGAATCAAAGG Glycogen storage disease, type IV, (p.Leu224Pro) GLYCOGEN STORAGE DISEASE IV, NONPROGRESSIVE HEPATIC 137852911 NM_000419.3(ITGA2B):c.641T>C CTGGTGCYTGGGGCTCCTGGCGG Glanzmann thrombasthenia (p.Leu214Pro) 137852948 NM_138694.3(PKHD1):c.10658T>C GAGCCCAYTGAAATACGCTCAGG Polycystic kidney disease, infantile type (p.Ile3553Thr) 137852964 NM_024960.4(PANK2):c.178T>C ATTGACYCAGTCGGATTCAATGG (p.Ser60Pro) 137853020 NM_006899.3(IDH3B):c.395T>C TGCGGCYGAGGTAGGTGGTCTGG, Retinitis pigmentosa 46 (p.Leu132Pro) GCGGCYGAGGTAGGTGGTCTGGG 137853249 NM_033500.2(HK1):c.1550T>C GACTTCTYGGCCCTGGATCTTGG, Hemolytic anemia due to hexokinase deficiency (p.Leu517Ser) TTCTYGGCCCTGGATCTTGGAGG 137853270 NM_000444.5(PHEX):c.1664T>C AGCYCCAGAAGCCTTTCTTTTGG Familial X-linked hypophosphatemic vitamin (p.Leu555Pro) D refractory rickets 137853325 NM_003639.4(IKBKG):c.1249T>C TGGAGYGCATTGAGTAGGGCCGG Hypohidrotic ectodermal dysplasia with immune (p.Cys417Arg) deficiency, Hyper-IgM immunodeficiency, X- linked, with hypohidrotic ectodermal dysplasia 28932769 NM_002055.4(GFAP):c.1055T>C GGACCYGCTCAATGTCAAGCTGG Alexander disease (p.Leu352Pro) 397507439 NM_002769.4(PRSS1):c.116T>C TACCAGGYGTCCCTGAATTCTGG Hereditary pancreatitis (p.Val39Ala) 387906446 NM_000132.3(F8):c.1729T>C AAAGAAYCTGTAGATCAAAGAGG Hereditary factor VIII deficiency disease (p.Ser577Pro) 387906482 NM_000133.3(F9):c.1031T>C ACGAACAYCTTCCTCAAATTTGG Hereditary factor LX deficiency disease (p.Ile344Thr) 387906508 NM_000131.4(F7):c.983T>C GACGTYCTCTGAGAGGACGCTGG Factor VII deficiency (p.Phe328Ser) 387906532 NM_001040113.1(MYH11):c.3791T>C GAAGCYGGAGGCGCAGGTGCAGG Aortic aneurysm, familial thoracic 4 (p.Leu1264Pro) 387906658 NM_002465.3(MYBPC1):c.2566T>C CAAACCYATATCCGCAGAGTTGG Distal arthrogryposis type 1B (p.Tyr856His) 387906701 NM_003491.3(NAA10):c.109T>C TGGCCTTYCCTGGCCCCAGGTGG, N-terminal acetyltransferase deficiency GGCCTTYCCTGGCCCCAGGTGGG GACTTCAYTGAGGACCAGGGTGG, 387906717 NM_000377.2(WAS):c.881T>C ACTTCAYTGAGGACCAGGGTGGG Severe congenital neutropenia X-linked (p.Ile294Thr) 387906809 NM_000287.3(PEX6):c.1601T>C CTTCYGGGCCGGGACCGTGATGG, Peroxisome biogenesis disorder 4B (p.Leu534Pro) TTCYGGGCCGGGACCGTGATGGG 387906965 NM_024513.3(FYC01):c.4127T>C CAGCCYGATCCCCATCACTGTGG Cataract, autosomal recessive congenital 2 (p.Leu1376Pro) 387906967 NM_006147.3(IRF6):c.65T>C GCCYCTACCCTGGGCTCATCTGG Van der Woude syndrome, Popliteal pterygium (p.Leu22Pro) syndrome 387906982 NM_025132.3(WDR19):c.20T>C TCTCACYGCTAGAAAAGACTTGG Asphyxiating thoracic dystrophy 5 (p.Leu7Pro) 387907072 NM_032446.2(MEGF10):c.2320T>C GGGCAGYGTACTTGCCGCACTGG Myopathy, areflexia, respiratory distress, and (p.Cys774Arg) dysphagia, early-onset, Myopathy, areflexia, respiratory distress, and dysphagia, early-onset, mild variant 137854499 NM_005502.3(ABCA1):c.6026T>C GAGTYCTTTGCCCTTTTGAGAGG Familial hypoalphalipoproteinemia (p.Phe2009Ser) 387907117 NM_000196.3(HSD11B2):c.1012T>C CCGCCGCYATTACCCCGGCCAGG, Apparent mineralocorticoid excess (p.Tyr338His) CGCCGCYATTACCCCGGCCAGGG 387907170 NM_004453.3(ETFDH):c.1130T>C CCAAAACYCACCTTTCCTGGTGG (p.Leu377Pro) 387907205 NM_033360.3(KRAS):c.211T>C GGACCAGYACATGAGGACTGGGG, Cardiofaciocutaneous syndrome 2 (p.Tyr71His) CCAGYACATGAGGACTGGGGAGG, CAGYACATGAGGACTGGGGAGGG 387907240 NM_024110.4(CARD14):c.467T>C CAGCAGCYGCAGGAGCACCTGGG Pityriasis rubra pilaris (p.Leu156Pro) 387907282 NM_152296.4(ATP1A3):c.2431T>C TGCCATCYCACTGGCGTACGAGG Alternating hemiplegia of childhood 2 (p.Ser811Pro) 387907361 NM_005120.2(MED12):c.3493T>C AGGACYCTGAGCCAGGGGCCCGG Ohdo syndrome, X-linked (p.Ser1165Pro) 28933970 NM_006194.3(PAX9):c.62T>C GGCCGCYGCCCAACGCCATCCGG Tooth agenesis, selective, 3 (p.Leu21Pro) 137854472 NM_000138.4(FBN1):c.3128A>G TGCACYTGCCGTGGGTGCAGAGG Cardiomyopathy, not specified (p.Lys1043Arg) 727504261 NM_000257.3(MYH7):c.2708A>G AGCGCYCCTCAGCATCTGCCAGG (p.Glu903Gly) 81002853 NM_000059.3(BRCA2):c.476- ACCACYGGGGGTAAAAAAAGGGG, Familial cancer of breast, Breast-ovarian 2A>G TACCACYGGGGGTAAAAAAAGGG, cancer, familial 2, Hereditary cancer-predisposing syndrome 119473032 NM_021020.3(LZTS1):c.355A>G CCCTYCTCGGAGCCCTGTAGAGG (p.Lys119Glu) 193922801 NM_000540.2(RYR1):c.7043A>G TTCYCCTCCACGCTCTCGCCTGG not provided (p.Glu2348Gly) 36210419 NM_000218.2(KCNQ1):c.652A>G GCCCCTYGGAGCCCACGCAGAGG Torsades de pointes, Cardiac arrhythmia (p.Lys218Glu) 121964989 NM_000108.4(DLD):c.1483A>G TTCTCYAAAAGCTTCTGATAAGG Maple syrup urine disease, type 3 (p.Arg495Gly) 28936669 NM_000095.2(COMP):c.1418A>G ATTGYCGTCGTCGTCGTCGCAGG (p.Asp473Gly) 28936696 NM_018488.2(TBX4):c.1592A>G GTACYGTAAGGAAGATTCTCGGG, Ischiopatellar dysplasia (p.Gln531Arg) GGTACYGTAAGGAAGATTCTCGG 121965077 NM_000137.2(FAH):c.1141A>G TCCYGGTCTGACCATTCCCCAGG Tyrosinemia type I (p.Arg381Gly) 794728203 NM_000138.4(FBND:c.3344A>G ACTCAYCAATATCTGCAAAATGG Thoracic aortic aneurysms and aortic (p.Asp1115Gly) dissections 786205436 NM_003002.3(SDHD):c.275A>G GAATAGYCCATCGCAGAGCAAGG Fatal infantile mitochondrial cardiomyopathy (p.Asp92Gly) 72551317 NM_000784.3(CYP27A1):c.776A>G AGTCCACYTGGGGAGGAAGGTGG Cholestanol storage disease (p.Lys259Arg) 786205687 NM_016218.2(POLK):c.1385A>G ATTCACAYTCTTCAACTTAATGG Malignant tumor of prostate (p.Asn462Ser) 794728280 NM_000138.4(FBN1):c.7916A>G TGTTCAYACTGGAAGCCGGCGGG, Thoracic aortic aneurysms and aortic (p.Tyr2639Cys) CTGTTCAYACTGGAAGCCGGCGG dissections 28937317 NM_000335.4(SCN5A):c.3971A>G GCAYTGACCACCACCTCAAGTGG Long QT syndrome 3, Congenital long QT (p.Asn1324Ser) syndrome 786205854 NM_144499.2(GNAT1):c.386A>G CGGAGYCCTTCCACAGCCGCTGG NIGHT BLINDNESS, (p.Asp129Gly) CONGENITAL 104893776 NM_000539.3(RHO):c.533A>G GGATGYACCTGAGGACAGGCAGG Retinitis pigmentosa 4 (p.TyrI78Cys) 28937590 NM_001257342.1(BCS1L):c.232A>G GACACYGAGGTGCTGAGTACGGG, GRACILE syndrome (p.Ser78Gly) CGACACYGAGGTGCTGAGTACGG 104893866 NM_000320.2(QDPR):c.449A>G TGCCGYACCCGATCATACCTGGG, Dihydropteridine reductase deficiency (p.Tyr150Cys) ATGCCGYACCCGATCATACCTGG 587776590 NM_015629.3(PRPF31):c.527+3A>G GACAYACCCCTGGGTGGTGGAGG, Retinitis pigmentosa 11 GCGGACAYACCCCTGGGTGGTGG 104894015 NM_000162.3(GCK):c.64IA>G GTAGYAGCAGGAGATCATCGTGG Hyperinsulinemic hypoglycemia familial 3 (p.Tyr214Cys) 202247823 NM_000532.4(PCCB):c.1606A>G ATATYTGCATGTTTTCTCCAAGG Propionic ac dem a (p.Asn536Asp) 104894199 NM_000073.2(CD3G):c.IA>G CCAYGTCAGTCTCTGTCCTCCGG Immunodeficiency 17 (p.Met1Val) 104894208 NM_001814.4(CTSC):c.857A>G CTCCYGAGGGCTTAGGATTGGGG, Papillon-Lef\xc3\xa8vre syndrome, Haim- (p.Gln286Arg) CCTCCYGAGGGCTTAGGATTGGG, Munk syndrome ACCTCCYGAGGGCTTAGGATTGG 104894211 NM_001814.4(CTSC):c.1040A>G TCCTACAYAGTGGTACTCAGAGG Papillon-Lenxc3\xa8vre (p.Tyr347Cys) syndrome, Periodontitis, 104894290 NM_000448.2(RAG1):c.2735A>G CTGYACTGGCAGAGGGATTCTGG Histiocytic medullary reticulosis (p.Tyr912Cys) 104894354 NM_000217.2(KCNA1):c.676A>G GCGYTTCCACGATGAAGAAGGGG, Episodic ataxia type 1 (p.T1u.226Ala) AGCGYTTCCACGATGAAGAAGGG, CAGCGYTTCCACGATGAAGAAGG 104894425 NM_014239.3(EIF2B2):c.638A>G AGTTGTCYCAATACCTGCTTTGG Leukoencephalopathy with vanishing bite (p.Glu213Gly) matter, Ovarioleukodystrophy 104894450 NM_000270.3(PNP):c.383A>G ATAYCTCCAACCTCAAACTTGGG, Purine-nucleoside phosphorylase deficiency (p.Asp128Gly) GATAYCTCCAACCTCAAACTTGG 147394623 NM_024887.3(DHDDS):c.124A>G GGCACTYCTTGGCATAGCGACGG Retinitis pigmentosa 59 (p.Lys42Glu) 60723330 NM_005557.3(KRT16):c.374A>G GCGGTCAYTGAGGTTCTGCATGG Pachyonychia congenita, type 1, Palmoplantar (p.Asn125Ser) keratoderma, nonepidermolytic, focal 104894634 NM_030665.3(RAI1):c.4685A>G CTGCTGCYGTCGTCGTCGCTTGG Smith-Magenis syndrome (p.Gln1562Arg) 104894730 NM_000363.4(TNNI3):c.532A>G CCTYCTTCACCTGCTTGAGGTGG, Familial restrictive cardiomyopathy 1 (p.Lys178Glu) CCTCCTYCTTCACCTGCTTGAGG 104894816 NM_002049.3(GATA1):c.653A>G GTCCTGYCCCTCCGCCACAGTGG GATA-1-related thrombocytopenia (p.Asp218Gly) with dyserythropoiesis 794726773 NM_001165963.1(SCN1A):c.1662+3 GTGCCAYACCTGGTGTGGGGAGG Severe myoclonic epilepsy in infancy A>G 104894861 NM_000202.6(IDS):c.404A>G AAAGACTYTTCCCACCGACATGG Mucopolysaccharidosis, MPS-II (p.Lys135Arg) 104894874 NM_000266.3(NDP):c.125A>G TGGYGCCTCATGCAGCGTCGAGG (p.His42Arg) 191205969 NM_002420.5(TRPM1):c.296T>C AAGCYCTTAATATCTGTGCATGG Congenital stationary night blindness, type 1C (p.Leu99Pro) 794727073 NM_019109.4(ALG1):c.1188- TAAACYGCAGAGAGAACCAAGGG, Congenital disorder of glycosylation type 1K 2A>G GTAAACYGCAGAGAGAACCAAG G 281875236 NM_001004334.3(GPR179):c.659A>G CCCACAYATCCATCTGCCTGCGG Congenital stationary night blindness, type 1E (p.Tyr220Cys) 28939094 NM_015915.4(ATL1):c.1222A>G CACCCAYCTTCTTCACCCCTCGG Spastic paraplegia 3 (p.Met408Val) 281875324 NM_005359.5(SMAD4):c.989A>G ATCCATTYCAAAGTAAGCAATGG Juvenile polyposis syndrome, Hereditary (p.Glu330Gly) cancer-predisposing syndrome 77173848 NM_000037.3(ANK1):c.- GGGCCYGGCCCGCACGTCACAGG Spherocytosis, type 1, autosomal recessive

150181226 NM_001159772.1(CANT1):c.671T>C CGTCYGTACGTGGGCGGCCTGGG, Desbuquois syndrome (p.Leu224Pro) GCGTCYGTACGTGGGCGGCCTGG 397514253 NM_000041.3(APOE):c.237- CGCCCYGCGGCCGAGAGGGCGGG, Familial type 3 hyperlipoprote nem a 2A>G GCGCCCYGCGGCCGAGAGGGCGG 397514348 NM_000060.3(BTD):c.278A>G GTTCAYAGATGTCAAGGTTCTGG Biotinidase deficiency (p.Tyr93Cys) 397514415 NM_000060.3(BTD):c.1313A>G GGCAYACAGCTCTTTGGATAAGG Biotinidase deficiency (p.Tyr438Cys) 397514501 NM_007171.3(POMT1):c.430A>G GAGCATYCTCTGTTTCAAAGAGG Limb-girdle muscular (p.Asn144Asp) dystrophy- 370382601 NM_174917.4(ACSF3):c.IA>G GGCAGCAYTGCACTGACAGGCGG not provided (P.Met1Val) 72554332 NM_000531.5(OTC):c.238A>G AAGGACTYCCCTTGCAATAAAGG Ornithine carbamoyltransferase deficiency (p.Lys80Glu) 397514599 NM_033109.4(PNPT1):c.1424A>G GACTYCAGATGTAACTCTTATGG Deafness, autosomal recessive 70 (p.Glu475Gly) 397514650 NM_000108.4(DLD):c.1444A>G GACTCYAGCTATATCTTCACAGG Maple syrup urine disease, type 3 (p.Arg482G1y) 397514675 NM_003156.3(STIMI):c.25IA>G TTCCACAYCCACATCACCATTGG Myopathy with tubular aggregates (p.Asp84G1y) 794728378 NM_000238.3(KCNH2):c.1913A>G ATCYTCTCTGAGTTGGTGTTGGG, Cardiac arrhythmia (p.Lys638Arg) GATCYTCTCTGAGTTGGTGTTGG 397514711 NM_002163.2(IRF8):c.238A>G AACCTCGYCTTCCAAGTGGCTGG Autosomal dominant CD11C+/CD1C+dendritic (p.Thr80Ala) cell deficiency 397514729 NM_000388.3(CASR):c.85A>G CCCCCTYCTTTTGGGCTCGCTGG Hypocalcemia, autosomal dominant 1, with (p.Lys29Glu) bartter syndrome 397514743 NM_022114.3(PRDM16):c.2447A>G GCCGCCGYTTTGGCTGGCACGGG Left ventricular noncompaction 8 (p.Asn816Ser) 397514757 NM_005689.2(ABCB6):c.508A>G TGGGCYGTTCCAAGACACCAGGG, Dyschromatosis universalis hereditaria 3 (p.Ser170Gly) GTGGGCYGTTCCAAGACACCAGG 28940313 NM_152443.2(RDH12):c.677A>G CACTGCGYAGGTGGTGACCCCGG Leber congenital amaurosis 13 (p.Tyr226Cys) 794728538 NM_000218.2(KCNQ1):c.1787A>G GTCTYCTACTCGGTTCAGGCGGG, Cardiac arrhythmia (p.Glu596Gly) TGTCTYCTACTCGGTTCAGGCGG 794728569 NM_000218.2(KCNQ1):c.605A>G AGGYCTGTGGAGTGCAGGAGAGG Cardiac arrhythmia (p.Asp202Gly) 794728573 NM 000218.2(KCNQ1):c.1515- GCCYGCAGTGGAGAGAGGAGAGG Cardiac arrhythmia 2A>G 370874727 NM_003494.3(DYSF):c.3349- CCGCCCYGGAGACACGAAGCTGG Limb-girdle muscular dystrophy, type 2B 2A>G 794728859 NM_198056.2(SCN5A):c.2788- ACCYGTCGAGATAATGGGTCAGG not provided 2A>G 794728887 NM_198056.2(SCN5A):c.4462A>G CCTCTGYCATGAAGATGTCCTGG not provided (p.Thr1488Ala) 28940878 NM_000372.4(TYR):c.125A>G CTCCTGYCCCCGCTCCACGGTGG Tyrosinase-negative oculocutaneous albinism (p.Asp42G1y) 397515420 NM_172107.2(KCNQ2):c.1636A>G GCAYGACACTGCAGGGGGGTGGG, Early infantile epileptic encephalopathy 7 (p.Met546Val) CGCAYGACACTGCAGGGGGGTGG, AACCGCAYGACACTGCAGGGGGG 397515428 NM_001410.2(MEGF8):c.7099A>G GACYCCCGTGAAATGATTCCCGG Carpenter syndrome 2 (p.Ser2367Gly) 143601447 NM_201631.3(TGM5):c.122T>C TCAACCYCACCCTGTACTTCAGG Peeling skin syndrome, acral type (p.Leu41Pro) 397515519 NM_000207.2(INS):c.*59A>G GGGCYTTATTCCATCTCTCTCGG Permanent neonatal diabetes mellitus 397515523 NM_000370.3(TTPA):c.191A>G CAGGYCCAGATCGAAATCCCGGG, Ataxia with vitamin E deficiency (p.Asp64G1y) CCAGGYCCAGATCGAAATCCCGG 397515891 NM_000256.3(MYBPC3):c.1224- TACTTGCYGTAGAACAGAAGGGG Familial hypertrophic cardiomyopathy 4, Cardiomyopathy 2A>G 397516082 NM_000256.3(MYBPC3):c.927- GTCCCYGTGTCCCGCAGTCTAGG Familial hypertrophic cardiomyopathy 2A>G 4, Cardiomyopathy 397516138 NM_000257.3(MYH7):c.2206A>G TATCAAYGAACTGTCCCTCAGGG, Familial hypertrophic cardiomyopathy (p.Ile736Val) CTATCAAYGAACTGTCCCTCAGG 1, Cardiomyopathy, not specified 1154510 NM_002150.2(HPD):c.97G>A ATGACGYGGCCTGAATCACAGGG, 4-Alpha-hydroxyphenylpyruvate hydroxylase (p.Ala33Thr) AATGACGYGGCCTGAATCACAGG deficiency 397516330 NM_000260.3(MY07A):c.6439- ATATCCYGGGGGAGCAGAAAGGG, Usher syndrome, type 1 2A>G GATATCCYGGGGGAGCAGAAAGG 72556271 NM_000531.5(OTC):c.482A>G CAGCCCAYTGATAATTGGGATGG not provided (p.Asn161Ser) 606231260 NM_023073.3(C5orf42):c.3290- ATCYATCAAATACAAAAATTTGG Orofaciodigital syndrome 6 2A>G 587777521 NM_004817.3(TJP2):c.1992- CAGCTCYGAGAAGAAACCACGGG, Progressive familial intrahepatic cholestasis 4 2A>G TCAGCTCYGAGAAGAAACCACGG 730880846 NM_000257.3(MYH7):c.617A>G CTTCYTGCTGCGGTCCCCAATGG Cardiomyopathy (p.Lys206Arg) 397517978 NM_206933.2(USH2A):c.12067- TTCCCYGTAAGAAAATTAACAGG Usher syndrome, type 2A, Retinitis pigmentosa 2A>G 39 606231409 NM_000216.2(ANOS1):c.1A>G GCACCAYGGCTGCGGGTCGAGGG, Kallmann syndrome 1 (p.Met1Val) GGCACCAYGGCTGCGGGTCGAGG 80356546 NM_003334.3(UBA1):c.1639A>G TGGCYTGTCACCCGGATATGTGG Arthrogryposis multiplex congenita, distal, (p.Ser547Gly) X- linked 80356584 NM_194248.2(OTOF):c.766- GACCYGCAGGCAGGAGAAGGGGG, Deafness, autosomal recessive 9 2A>G TGACCYGCAGGCAGGAGAAGGGG, CTGACCYGCAGGCAGGAGAAGGG, GCTGACCYGCAGGCAGGAGAAGG 730880930 NM_000257.3(MYH7):c.1615A>G GGAACAYGCACTCCTCTTCCAGG Cardiomyopathy (p.Met539Val) 118203947 NM_013319.2(UBIAD1):c.355A>G TCCYGTCATCACTCTTTTTGTGG Schnyder crystalline conical dystrophy (p.Arg119Gly) 60171927 NM_000526.4(KRT14):c.368A>G GCGGTCAYTGAGGTTCTGCATGG Epidermolysis bullosa herpetiformis, (p.Asn123Ser) Dowling- Meara 199422248 NM_001363.4(DKC1):c.941A>G AATCYTGGCCCCATAGCAGATGG Dyskeratosis congenita X-linked (p.Lys314Arg) 72558467 NM_000531.5(OTC):c.929A>G TCCACTYCTTCTGGCTTTCTGGG, not provided (p Glu310Gly) ATCCACTYCTTCTGGCTTTCTGG 72558478 NM_000531.5(OTC):c.988A>G ACTTTCYGTTTTCTGCCTCTGGG, not provided (p.Arg330G1y) CACTTTCYGTTTTCTGCCTCTGG 118204455 NM_000505.3(F12):c.158A>G GGTGGYACTGGAAGGGGAAGTGG (p.Tyr53Cys) 80357477 NM_007294.3(BRCA1):c TTGYCCTCTGTCCAGGCATCTGG Familial cancer of breast, Breast-ovarian (p.Asp1818Gly) cancer, familial 1 121907908 NM_024426.4(WT1):c.1021A>G CGCYCTCGTACCCTGTGCTGTGG Mesothelioma (p.Ser341Gly) 121907926 NM_000280.4(PAX6):c.1171A>G GTGGYGCCCGAGGTGCCCATTGG Optic nerve aplasia, bilateral (p.Thr391Ala) 121908023 NM_024740.2(ALG9):c.860A>G TTAYACAAAACAATGTTGAGTGG Congenital disorder of glycosylation type 1L (p.Tyr287Cys) 121908148 NM_001243133.1(NLRP3):c.1880A>G ACAATYCCAGCTGGCTGGGCTGG Familial cold urticaria (p.Glu627Gly) 121908166 NM_006492.2(ALX3):c.608A>G CGGYTCTGGAACCAGACCTGGGG, Frontonasal dysplasia (p.Asn203Ser) GCGGYTCTGGAACCAGACCTGGG, TGCGGYTCTGGAACCAGACCTGG 121908184 NM_020451.2(SEPN1):c.1A>G CCCAYGGCTGCGGCTGGCGGCGG, Eichsfeld type congenital muscular dystrophy (p.Met1Val) CGGCCCAYGGCTGCGGCTGGCGG 121908258 NM_130468.3(CHST14):c.878A>G AAGTCAYAGTGCACGGCACAAGG Ehlers-Danlos syndrome, musculocontractural (p.Tyr293Cys) type 121908383 NM_001128425.1(MUTYH):c.1241A>G AAGCYGCTCTGAGGGCTCCCAGG Neoplasm of stomach (p.Gln414Arg) 121908580 NM_004328.4(BCS1L):c.148A>G GTGYGATCATGTAATGGCGCCGG Mitochondrial complex III deficiency (p.Thr50Ala) 121908584 NM_016417.2(GLRX5):c.294A>G CCTGACCYTGTCGGAGCTCCGGG Anemia, sideroblastic, pyridoxine-refractory, (p.Gln98=) autosomal recessive 121908635 NM_022817.2(PER2):c.1984A>G GCCACACYCTCTGCCTTGCCCGG Advanced sleep phase syndrome, familial (p.Ser662Gly) 121908655 NM_003839.3(TNERSF11A):c.508A>G GGGTCYGCATTTGTCCGTGGAGG Osteopetrosis autosomal recessive 7 (p.Arg170Gly) 29001653 NM_000539.3(RHO):c.886A>G CGCTCTYGGCAAAGAACGCTGGG, Retinitis pigmentosa 4 (p.Lys296Glu) GCGCTCTYGGCAAAGAACGCTGG 56307355 NM_006502.2(POLH):c.1603A>G AGACTTTYCTGCTTAAAGAAGGG Xeroderma pigmentosum, variant type (p.Lys535Glu) 121908919 NM_002977.3(SCN9A):c.1964A>G CCTTTTCYTGTGTATTTGATTGG Generalized epilepsy with febrile seizures plus, (p.Lys655Arg) type 7 not specified 121908939 NM_006892.3(DNMT3B):c.2450A>G GACACGYCTGTGTAGTGCACAGG Centromeric instability of chromosomes 1,9 and (p.Asp817Gly) 16 and immunodeficiency 121909088 NM_001005360.2(DNM2):c.1684A>G ACTYCTTCTCTTTCTCCTGAGGG, Charcot-Marie-Tooth disease, dominant (p.Lys562Glu) TACTYCTTCTCTTTCTCCTGAGG intermediate b, with neutropenia 120074112 NM_000483.4(APOC2):c.1A>G GCCCAYAGTGTCCAGAGACCTGG Apolipoprotein C2 deficiency (p.Met1Val) 121909239 NM_000314.6(PTEN):c.755A>G ATAYCACCACACACAGGTAACGG Macrocephaly/autism syndrome (p.Asp252G1y) 121909251 NM_198217.2(ING1):c.515A>G TGGYTGCACAGACAGTACGTGGG, Squamous cell carcinoma of the head and neck (p.Asn172Ser) CTGGYTGCACAGACAGTACGTGG 121909396 NM_001174089.1(SLC4A11):c.2518A>G GATCAYCTTCATGTAGGGCAGGG, Corneal dystrophy and perceptive deafness (p.Met840Val) AGATCAYCTTCATGTAGGGCAGG 121909533 NM_000034.3(ALD0A):c.386A>G CCAYCCAACCCTAAGAGAAGAGG HNSHA due to aldolase A deficiency (p.Asp129Gly) 128627255 NM_004006.2(DMD):c.835A>G TGACCGYGATCTGCAGAGAAGGG, Dilated cardiomyopathy 3B (p.Thr279Ala) CTGACCGYGATCTGCAGAGAAGG 116929575 NM_001085.4(SERPINA3):c.1240A>G GCTCAYGAAGAAGATGTTCTGGG, (p.Met414Val) TGCTCAYGAAGAAGATGTTCTGG 61748392 NM_004992.3(MECP2):c.410A>G CAACYCCACTTTAGAGCGAAAGG Mental retardation, X-linked, syndromic 13 (p.Glu137Gly) 61748906 NM_001005741.2(GBA):c.667T>C CCCACTYGGCTCAAGACCAATGG Gaucher disease, type 1 (p.Trp223Arg) 199473024 NM_000238.3(KCNH2):c.3118A>G CTGCYCTCCACGTCGCCCCGGGG, Sudden infant death syndrome (p.Ser1040Gly) CCTGCYCTCCACGTCGCCCCGGG, GCCTGCYCTCCACGTCGCCCCGG 794728365 NM_000238.3(KCNH2):c.1129- GGACCYGCACCCGGGGAAGGCGG Cardiac arrhythmia

72556293 NM_000531.5(OTC):c.548A>G AGAGCTAYAGTGTTCCTAAAAGG not provided (p.Tyr183Cys) 111033244 NM_000441.1(SLC26A4):c.1151A>G TGAATYCCTAAGGAAGAGACTGG Pendred syndrome, Enlarged vestibular (p.Glu384Gly) aqueduct syndrome 111033415 NM_000260.3(MY07A):c.1344- AGCYGCAGGGGCACAGGGATGGG, Usher syndrome, type 1 2A>G AAGCYGCAGGGGCACAGGGATGG 121912439 NM_000454.4(SOD1):c.302A>G AGAATCTYCAATAGACACATCGG Amyotrophic lateral sclerosis type 1 (p.Glu101Gly) 111033567 NM_002769.4(PRSS1):c.68A>G ATCYTGTCATCATCATCAAAGGG, Hereditarypancreatitis (p.Lys23Arg) GATCYTGTCATCATCATCAAAG G 121912565 NM_000901.4(NR3C2):c.2327A>G TCATCYGTTTGCCTGCTAAGCGG Pseudohypoaldosteronism type 1 autosomal (p.Gln776Arg) dominant 121912574 NM_000901.4(NR3C2):c.2915A>G CCGACYCCACCTTGGGCAGCTGG Pseudohypoaldosteronism type 1 autosomal (p.Glu972Gly) dominant 121912589 NM_001173464.1(KIF21A):c.2839A>G ATTCAYATCTGCCTCCATGTTGG Fibrosis of extraocular muscles, congenital, 1 (p.Met947Val) 111033661 NM_000155.3(GALT):c.253- ATTCACCYACCGACAAGGATAGG Deficiency of UDPglucose-hexose-1-phosphate 2A>G uridylyltransferase 111033669 NM_000155.3(GALT):c.290A>G GAAGTCGYTGTCAAACAGGAAGG Deficiency of UDPglucose-hexose-1-phosphate (p.Asn97Ser) uridylyltransferase 111033682 NM_000155.3(GALT):c.379A>G TGACCTYACTGGGTGGTGACGGG, Deficiency of UDPglucose-hexose-1-phosphate (p.Lys127Glu) ATGACCTYACTGGGTGGTGACGG uridylyltransferase 111033786 NM_000155.3(GALT):c.950A>G CAGCYGCCAATGGTTCCAGTTGG Deficiency of UDPglucose-hexose-1-phosphate (p.Gln317Arg) uridylyltransferase 121912765 NM_001202.3(BMP4):c.278A>G CCTCCYCCCCAGACTGAAGCCGG Microphthalmia syndromic 6 (p.Glu93Gly) 121912856 NM_000094.3(COL7A1):c.425A>G CACCYTGGGGACACCAGGTCGGG, Epidermolysis bullosa dystrophica inversa, (p.Lys142Arg) TCACCYTGGGGACACCAGGTCGG autosomal recessive 199474715 NM_152263.3(TPM3):c.505A>G CCAACTYACGAGCCACCTACAGG Congenital myopathy with fiber (p.Lys169Glu) type disproportion 199474718 NM_152263.3(TPM3):c.733A>G ATCYCTCAGCAAACTCAGCACGG Congenital myopathy with fiber (p.Arg245Gly) type disproportion 121912895 NM_001844.4(COL2A1):c.2974A>G CCTCYCTCACCACGTTGCCCAGG Spondyloepimetaphyseal dysplasia Strudwick (p.Arg992Gly) type 121913074 NM_000129.3(F13A1):c.851A>G ATAGGCAYAGATATTGTCCCAGG Factor xiii, a subunit, deficiency of (p.Tyr284Cys) 121913145 NM_000208.2(INSR):c.707A>G GCTGYGGCAACAGAGGCCTTCGG Leprechaunism syndrome (p.His236Arg) 312262745 NM_025137.3(SPG11):c.2608A>G ACTTAYCCTGGGGAGAAGGATGG Spastic paraplegia 11, autosomal recessive (p.Ile870Val) 121913682 NM_000222.2(KIT):c.2459A>G AGAAYCATTCTTGATGTCTCTGG Mast cell disease, systemic (p.Asp820Gly) 587776757 NM_000151.3(G6PC):c.230+4A>G GTTCYTACCACTTAAAGACGAGG Glycogen storage disease type 1A 61752063 NM_000330.3(RS1):c.286T>C TTCTTCGYGGACTGCAAACAAGG Juvenile retinoschisis (p.Trp96Arg) 367543065 NM_024549.5(TCTN1):c.221- AGCAACYGCAGAAAAAAGAGGGG, Joubert syndrome 13 2A>G CAGCAACYGCAGAAAAAAGAGG G 5030773 NM_000894.2(LHB):c.221A>G CCACCYGAGGCAGGGGCGGCAGG Isolated lutropin deficiency (p.Gln74Arg) 199476092 NM_000264.3(PTCH1):c.2479A>G CGTTACYGAAACTCCTGTGTAGG Gorlin syndrome, Holoprosencephaly 7, not (p.Ser827Gly) specified 398123158 NM_000117.2(EMD):c.450- CGTTCCCYGAGGCAAAAGAGGGG not provided 2A>G 199476103 RMRP:n.71A>G ACTTYCCCCTAGGCGGAAAGGGG, Metaphyseal chondrodysplasia, McKusick type, GACTTYCCCCTAGGCGGAAAGGG, Metaphyseal dysplasia without hypotrichosis GGACTTYCCCCTAGGCGGAAAGG 5030856 NM_000277.1(PAH):c.1169A>G CTCYCTGCCACGTAATACAGGGG, Phenylketonuria, Hyperphenylalaninemia, non- (p.Glu390Gly) ACTCYCTGCCACGTAATACAGGG, Pku AACTCYCTGCCACGTAATACAGG 5030860 NM_000277.1(PAH):c.1241A>G GGGTCGYAGCGAACTGAGAAGGG, Phenylketonuria, Hyperphenylalaninem a non- (p.Tyr414Cys) TGGGTCGYAGCGAACTGAGAAGG Pku 587777055 NM_020988.2(GNAO1):c.521A>G GGATGYCCTGCTCGGTGGGCTGG Early infantile epileptic encephalopathy 17 (p.Asp174Gly) 587777223 NM_024301.4(FKRP):c.1A>G CCGCAYGGGGCCGAAGTCTGGGG, Congenital muscular dystrophy-dystroglycanopathy (p.Met1Val) GCCGCAYGGGGCCGAAGTCTGGG, with brain and eye anomalies type AS AGCCGCAYGGGGCCGAAGTCTGG 587777479 NM_003108.3(S0X11):c.347A>G GTACTTGYAGTCGGGGTAGTCGG Mental retardation, autosomal dominant 27 (p.Tyr116Cys) 587777496 NM_020435.3(GJC2):c.-170A>G TTGYTCCCCCCTCGGCCTCAGGG, Leukodystrophy, hypomyelinating, 2 ATTGYTCCCCCCTCGGCCTCAGG 587777507 NM_022552.4(DNMT3A):c.1943T>C CTCCYGGTGCTGAAGGACTTGGG, Tatton-Brown-rahman syndrome (p.Leu648Pro) GCTCCYGGTGCTGAAGGACTTGG 587777557 NM_018400.3(SCN3B):c.482T>C AATCAYGATGTACATCCTTCTGG Atrial fibrillation, familial, 16 (p.Met161Thr) 587777569 NM_001030001.2(RPS29):c.149T>C GATAYCGGTTTCATTAAGGTAGG Diamond-Blackfan anemia 13 (p.Ile50Thr) 587777657 NM_153334.6(SCARF2):c.190T>C CCACGYGCTGCGCTGGCTGGAGG Marden Walker like syndrome (p.Cys64Arg) 587777689 NM_005726.5(TSFM):c.57+4A>G ACTTCYCACCGGGTAGCTCCCGG Combined oxidative phosphorylation deficiency 3 796052005 NM_000255.3(MUT):c.329A>G GCAYACTGGCGGATGGTCCAGGG, not provided (p.Tyr110Cys) AGCAYACTGGCGGATGGTCCAGG 587777809 NM_144596.3(TTC8):c.115- GTTCCYGGAAAGCATTAAGAAGG Retinitis pigmentosa 51 2A>G 587777878 NM_000166.5(GJB1):c.580A>G TAGCAYGAAGACGGTGAAGACGG X-linked hereditary motor and sensory (p.Met194Val) neuropathy 74315420 NM_001029871.3(RSPO4):c.194A>G CGTACYGGCGGATGCCTTCCCGG Anonychia (p.Gln65Arg) 180177219 NM_000030.2(AGXT):c.424-2A>G AGGCCCYGAGGAAGCAGGGACGG Primary hyperoxaluria, type I (p.Gly_142Gln145del) 367610201 NM_002693.2(POLG):c.1808T>C CTCAYGGCACTTACCTGGGATGG not provided (p.Met603Thr) 180177319 NM_012203.1(GRHPR):c.84- TCACAGCYGCGGGGAAAGGGAGG Primary hyperoxaluria, type II 2A>G 796052068 NM_000030.2(AGXT):c.777- GGTACCYGGAAGACACGAGGGGG, Primary hyperoxaluria, type I 2A>G TGGTACCYGGAAGACACGAGGGG 61754010 NM_000552.3(VWF):c.1583A>G TGCCAYTGTAATTCCCACACAGG von Willebrand disease, type 2a (p.Asn528Ser) 587778866 NM_000321.2(RB1):c.1927A>G ATTYCAATGGCTTCTGGGTCTGG Retinoblastoma (p.Lys643Glu) 74435397 NM_006331.7(EMG1):c.257A>G ATAYCTGGCCGCGCTTCCCCAGG Bowen-Conradi syndrome (p.Asp86G1y) 796052527 NM_000156.5(GAMT):c.1A>G CGCTCAYGCTGCAGGCTGGACGG not provided (p.Met1Val) 796052637 NM_172107.2(KCNQ2):c.848A>G GTACYTGTCCCCGTAGCCAATGG not provided (p.Lys283Arg) 724159963 NM_032228.5(FARH:c.1094A>G GATAYCATACAGGAATGCTGGGG, Peroxisomal fatty acyl-coa reductase 1 disorder (p.Asp365Gly) AGATAYCATACAGGAATGCTGGG, TAGATAYCATACAGGAATGCTGG 587779722 NM_000090.3(COL3A1):c.1762-2A>G CACCCYAAAGAAGAAGTGGTCGG Ehlers-Danlos syndrome, type 4 (p.Gly588_Gln605del) 118192102 m.8296A>G TTTACAGYGGGCTCTAGAGGGGG Diabetes-deafness syndrome maternally transmitted 727502787 NM_001077494.3(NEKB2):c.2594A>G CTGYCTTCCTTCACCTCTGCTGG Common variable immunodeficiency 10 (p.Asp865Gly) 727503036 NM_000117.2(EMD):c.266- AGCCYTGGGAAGGGGGGCAGCGG Emery-Dreifuss muscular dystrophy 1, X-linked 2A>G 690016544 NM_005861.3(STUB1):c.194A>G GGCCCGGYTGGTGTAATACACGG Spinocerebellar ataxia, autosomal recessive 16 (p.Asn65Ser) 690016554 NM_005211.3(CSF1R):c.2655- GTATCYGGGAGATAGGACAGAGG Hereditary diffuse leukoencephalopathy with 2A>G spheroids 118192185 NM_172107.2(KCNQ2):c.1A>G GCACCAYGGTGCCTGGCGGGAGG Benign familial neonatal seizures 1 (p.Met1Val) 121917869 NM_012064.3(MIP):c.401A>G AGATCYCCACTGTGGTTGCCTGG Cataract 15, multiple types (p.Glu134Gly) 121918014 NM_000478.4(ALPL):c.1250A>G AGGCCCAYTGCCATACAGGATGG Infantile hypophosphatasia (p.Asn417Ser) 121918036 NM_000174.4(GP9):c.110A>G GCAGYCCACCCACAGCCCCATGG Bernard-Soulier syndrome type C (p.Asp37G1y) 121918089 NM_000371.3(TTR):c.379A>G CGGCAAYGGTGTAGCGGCGGGGG, Amyloidogenic transthyretin amyloidosis (p.Ile127Val) GCGGCAAYGGTGTAGCGGCGGGG 121918121 NM_000823.3(GHRHR):c.985A>G CGACTYGGAGAGACGCCTGCAGG Isolated growth hormone deficiency type 1B (p.Lys329Glu) 121918333 NM_015335.4(MED13L):c.6068A>G ATATCAYCTAGAGGGAAGGGGGG, Transposition of great arteries (p.Asp2023Gly) CATATCAYCTAGAGGGAAGGGGG 121918605 NM_001035.2(RYR2):c.12602A>G CGCCAGCYGCATTTCAAAGATGG Catecholaminergic polymorphic (p.Gln4201Arg) ventricular tachycardia 587781262 NM_002764.3(PRPS1):c.343A>G TAGCAYATTTGCAACAAGCTTGG Charcot-Marie-Tooth disease, X-linked (p.Met115Val) recessive, type 5, Deafness, high-frequency sensorineural, X-linked 121918608 NM_001161766.1(AHCY):c.344A>G GCGGGYACTTGGTGTGGATGAGG Hypermethioninemia with s-adenosylhomocysteine (p.Tyr115Cys) hydrolase deficiency 121918613 NM_000702.3(ATP1A2):c.1033A>G CTGYCAGGGTCAGGCACACCTGG Familial hemiplegic migraine type 2 (p.Thr345Ala) 587781339 NM_000535.5(PMS2):c.904- GCAGACCYGCACAAAATACAAGG Hereditary cancer-predisposing syndrome 2A>G 121918691 NM_001128177.1(THRB):c.1324A>G CTTCAYGTGCAGGAAGCGGCTGG Thyroid hormone resistance, generalized, (p.Met442Val) autosomal dominant 121918692 NM_001128177.1(THRB):c.1327A>G CCACCTYCATGTGCAGGAAGCGG Thyroid hormone resistance, generalized, (p.Lys443Glu) autosomal dominant 727504333 NM_000256.3(MYBPC3):c.2906- CCGTTCYGTGGGTATAGAGTGGG, Familial hypertrophic cardiomyopathy 4 2A>G GCCGTTCYGTGGGTATAGAGTGG 730880805 NM_006204.3(PDE6C):c.1483- CTTTCYGTTGAAATAAGGATGGG, Achromatopsia 5 2A>G TCTTTCYGTTGAAATAAGGATGG 281860296 NM_000551.3(VHL):c.586A>T GGTCTTYCTGCACATTTGGGTGG Von Hippel-Lindau syndrome (p.Lys196Ter) 730880444 NM_000169.2(GLA):c.370- GTGAACCYGAAATGAGAGGGAGG not provided 2A>G 756328339 NM_000256.3(MYBPC3):c.1227- GTACCYGGGTGGGGGCCGCAGGG, Familial hypertrophic cardiomyopathy 2A>G TGTACCYGGGTGGGGGCCGCAGG 4, Cardiomyopathy 267606643 NM_013411.4(AK2):c.494A>G TCAYCTTTCATGGGCTCTTTTGG Reticular dysgenesis (p.Asp165Gly) 267606705 NM_005188.3(CBL):c.1144A>G TATTTYACATAGTTGGAATGTGG Noonan syndrome-like disorder with or without (p.Lys382Glu) juvenile myelomonocytic leukemia 62642934 NM_000277.1(PAH):c.916A>G GGCCAAYTTCCTGTAATTGGGGG, Phenylketonuria, Hyperphenylalaninemia, non- (p.Ile306Val) AGGCCAAYTTCCTGTAATTGGGG Pku 267606782 NM_000117.2(EMD):c.1A>G TCCAYGGCGGGTGCGGGCTCAGG Emery-Dreifuss muscular dystrophy, X-linked (p.Met1Val) 267606820 NM_014053.3(FLVCR1):c.361A>G AGGCGTYGACCAGCGAGTACAGG Posterior column ataxia with (p.Asn121Asp) retinitis pigmentosa

indicates data missing or illegible when filed

In some embodiments, any of the base editors provided herein may be used to treat a disease or disorder. For example, any base editors provided herein may be used to correct one or more mutations associated with any of the diseases or disorders provided herein. Exemplary diseases or disorders that may be treated include, without limitation, 3-Methylglutaconic aciduria type 2, 46, XY gonadal dysgenesis, 4-Alpha-hydroxyphenylpyruvate hydroxylase deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, achromatopsia, Acid-labile subunit deficiency, Acrodysostosis, acroerythrokeratoderma, ACTH resistance, ACTH-independent macronodular adrenal hyperplasia, Activated PI3K-delta syndrome, Acute intermittent porphyria, Acute myeloid leukemia, Adams-Oliver syndrome 1/5/6, Adenylosuccinate lyase deficiency, Adrenoleukodystrophy, Adult neuronal ceroid lipofuscinosis, Adult onset ataxia with oculomotor apraxia, Advanced sleep phase syndrome, Age-related macular degeneration, Alagille syndrome, Alexander disease, Allan-Herndon-Dudley syndrome, Alport syndrome, X-linked recessive, Alternating hemiplegia of childhood, Alveolar capillary dysplasia with misalignment of pulmonary veins, Amelogenesis imperfecta, Amyloidogenic transthyretin amyloidosis, Amyotrophic lateral sclerosis, Anemia (nonspherocytic hemolytic, due to G6PD deficiency), Anemia (sideroblastic, pyridoxine-refractory, autosomal recessive), Anonychia, Antithrombin III deficiency, Aortic aneurysm, Aplastic anemia, Apolipoprotein C2 deficiency, Apparent mineralocorticoid excess, Aromatase deficiency, Arrhythmogenic right ventricular cardiomyopathy, Familial hypertrophic cardiomyopathy, Hypertrophic cardiomyopathy, Arthrogryposis multiplex congenital, Aspartylglycosaminuria, Asphyxiating thoracic dystrophy, Ataxia with vitamin E deficiency, Ataxia (spastic), Atrial fibrillation, Atrial septal defect, atypical hemolytic-uremic syndrome, autosomal dominant CD11C+/CD1C+ dendritic cell deficiency, Autosomal dominant progressive external ophthalmoplegia with mitochondrial DNA deletions, Baraitser-Winter syndrome, Bartter syndrome, Basa ganglia calcification, Beckwith-Wiedemann syndrome, Benign familial neonatal seizures, Benign scapuloperoneal muscular dystrophy, Bernard Soulier syndrome, Beta thalassemia intermedia, Beta-D-mannosidosis, Bietti crystalline corneoretinal dystrophy, Bile acid malabsorption, Biotinidase deficiency, Borjeson-Forssman-Lehmann syndrome, Boucher Neuhauser syndrome, Bowen-Conradi syndrome, Brachydactyly, Brown-Vialetto-Van laere syndrome, Brugada syndrome, Cardiac arrhythmia, Cardiofaciocutaneous syndrome, Cardiomyopathy, Carnevale syndrome, Carnitine palmitoyltransferase II deficiency, Carpenter syndrome, Cataract, Catecholaminergic polymorphic ventricular tachycardia, Central core disease, Centromeric instability of chromosomes 1,9 and 16 and immunodeficiency, Cerebral autosomal dominant arteriopathy, Cerebro-oculo-facio-skeletal syndrome, Ceroid lipofuscinosis, Charcot-Marie-Tooth disease, Cholestanol storage disease, Chondrocalcinosis, Chondrodysplasia, Chronic progressive multiple sclerosis, Coenzyme Q10 deficiency, Cohen syndrome, Combined deficiency of factor V and factor VIII, Combined immunodeficiency, Combined oxidative phosphorylation deficiency, Combined partial 17-alpha-hydroxylase/17,20-lyase deficiency, Complement factor d deficiency, Complete combined 17-alpha-hydroxylase/17,20-lyase deficiency, Cone-rod dystrophy, Congenital contractural arachnodactyly, Congenital disorder of glycosylation, Congenital lipomatous overgrowth, Neoplasm of ovary, PIK3CA Related Overgrowth Spectrum, Congenital long QT syndrome, Congenital muscular dystrophy, Congenital muscular hypertrophy-cerebral syndrome, Congenital myasthenic syndrome, Congenital myopathy with fiber type disproportion, Eichsfeld type congenital muscular dystrophy, Congenital stationary night blindness, Corneal dystrophy, Cornelia de Lange syndrome, Craniometaphyseal dysplasia, Crigler Najjar syndrome, Crouzon syndrome, Cutis laxa with osteodystrophy, Cyanosis, Cystic fibrosis, Cystinosis, Cytochrome-c oxidase deficiency, Mitochondrial complex I deficiency, D-2-hydroxyglutaric aciduria, Danon disease, Deafness with labyrinthine aplasia microtia and microdontia (LAMM), Deafness, Deficiency of acetyl-CoA acetyltransferase, Deficiency of ferroxidase, Deficiency of UDPglucose-hexose-1-phosphate uridylyltransferase, Dejerine-Sottas disease, Desbuquois syndrome, DFNA, Diabetes mellitus type 2, Diabetes-deafness syndrome, Diamond-Blackfan anemia, Diastrophic dysplasia, Dihydropteridine reductase deficiency, Dihydropyrimidinase deficiency, Dilated cardiomyopathy, Disseminated atypical mycobacterial infection, Distal arthrogryposis, Distal hereditary motor neuronopathy, Donnai Barrow syndrome, Duchenne muscular dystrophy, Becker muscular dystrophy, Dyschromatosis universalis hereditaria, Dyskeratosis congenital, Dystonia, Early infantile epileptic encephalopathy, Ehlers-Danlos syndrome, Eichsfeld type congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, Enamel-renal syndrome, Epidermolysis bullosa dystrophica inversa, Epidermolysis bullosa herpetiformis, Epilepsy, Episodic ataxia, Erythrokeratodermia variabilis, Erythropoietic protoporphyria, Exercise intolerance, Exudative vitreoretinopathy, Fabry disease, Factor V deficiency, Factor VII deficiency, Factor xiii deficiency, Familial adenomatous polyposis, breast cancer, ovarian cancer, cold urticarial, chronic infantile neurological, cutaneous and articular syndrome, hemiplegic migraine, hypercholesterolemia, hypertrophic cardiomyopathy, hypoalphalipoproteinemia, hypokalemia-hypomagnesemia, juvenile gout, hyperlipoproteinemia, visceral amyloidosis, hypophosphatemic vitamin D refractory rickets, FG syndrome, Fibrosis of extraocular muscles, Finnish congenital nephrotic syndrome, focal epilepsy, Focal segmental glomerulosclerosis, Frontonasal dysplasia, Frontotemporal dementia, Fructose-biphosphatase deficiency, Gamstorp-Wohlfart syndrome, Ganglioside sialidase deficiency, GATA-1-related thrombocytopenia, Gaucher disease, Giant axonal neuropathy, Glanzmann thrombasthenia, Glomerulocystic kidney disease, Glomerulopathy, Glucocorticoid resistance, Glucose-6-phosphate transport defect, Glutaric aciduria, Glycogen storage disease, Gorlin syndrome, Holoprosencephaly, GRACILE syndrome, Haemorrhagic telangiectasia, Hemochromatosis, Hemoglobin H disease, Hemolytic anemia, Hemophagocytic lymphohistiocytosis, Carcinoma of colon, Myhre syndrome, leukoencephalopathy, Hereditary factor IX deficiency disease, Hereditary factor VIII deficiency disease, Hereditary factor XI deficiency disease, Hereditary fructosuria, Hereditary Nonpolyposis Colorectal Neoplasm, Hereditary pancreatitis, Hereditary pyropoikilocytosis, Elliptocytosis, Heterotaxy, Heterotopia, Histiocytic medullary reticulosis, Histiocytosis-lymphadenopathy plus syndrome, HNSHA due to aldolase A deficiency, Holocarboxylase synthetase deficiency, Homocysteinemia, Howel-Evans syndrome, Hydatidiform mole, Hypercalciuric hypercalcemia, Hyperimmunoglobulin D, Mevalonic aciduria, Hyperinsulinemic hypoglycemia, Hyperkalemic Periodic Paralysis, Paramyotonia congenita of von Eulenburg, Hyperlipoproteinemia, Hypermanganesemia, Hypermethioninemia, Hyperphosphatasemia, Hypertension, hypomagnesemia, Hypobetalipoproteinemia, Hypocalcemia, Hypogonadotropic hypogonadism, Hypogonadotropic hypogonadism, Hypohidrotic ectodermal dysplasia, Hyper-IgM immunodeficiency, Hypohidrotic X-linked ectodermal dysplasia, Hypomagnesemia, Hypoparathyroidism, Idiopathic fibrosing alveolitis, Immunodeficiency, Immunoglobulin A deficiency, Infantile hypophosphatasia, Infantile Parkinsonism-dystonia, Insulin-dependent diabetes mellitus, Intermediate maple syrup urine disease, Ischiopatellar dysplasia, Islet cell hyperplasia, Isolated growth hormone deficiency, Isolated lutropin deficiency, Isovaleric acidemia, Joubert syndrome, Juvenile polyposis syndrome, Juvenile retinoschisis, Kallmann syndrome, Kartagener syndrome, Kugelberg-Welander disease, Lattice corneal dystrophy, Leber congenital amaurosis, Leber optic atrophy, Left ventricular noncompaction, Leigh disease, Mitochondrial complex I deficiency, Leprechaunism syndrome, Arthrogryposis, Anterior horn cell disease, Leukocyte adhesion deficiency, Leukodystrophy, Leukoencephalopathy, Ovarioleukodystrophy, L-ferritin deficiency, Li-Fraumeni syndrome, Limb-girdle muscular dystrophy-dystroglycanopathy, Loeys-Dietz syndrome, Long QT syndrome, Macrocephaly/autism syndrome, Macular corneal dystrophy, Macular dystrophy, Malignant hyperthermia susceptibility, Malignant tumor of prostate, Maple syrup urine disease, Marden Walker like syndrome, Marfan syndrome, Marie Unna hereditary hypotrichosis, Mast cell disease, Meconium ileus, Medium-chain acyl-coenzyme A dehydrogenase deficiency, Melnick-Fraser syndrome, Mental retardation, Merosin deficient congenital muscular dystrophy, Mesothelioma, Metachromatic leukodystrophy, Metaphyseal chondrodysplasia, Methemoglobinemia, methylmalonic aciduria, homocystinuria, Microcephaly, chorioretinopathy, lymphedema, Microphthalmia, Mild non-PKU hyperphenylalanemia, Mitchell-Riley syndrome, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency, Mitochondrial complex I deficiency, Mitochondrial complex III deficiency, Mitochondrial myopathy, Mucolipidosis III, Mucopolysaccharidosis, Multiple sulfatase deficiency, Myasthenic syndrome, Mycobacterium tuberculosis, Myeloperoxidase deficiency, Myhre syndrome, Myoclonic epilepsy, Myofibrillar myopathy, Myoglobinuria, Myopathy, Myopia, Myotonia congenital, Navajo neurohepatopathy, Nemaline myopathy, Neoplasm of stomach, Nephrogenic diabetes insipidus, Nephronophthisis, Nephrotic syndrome, Neurofibromatosis, Neutral lipid storage disease, Niemann-Pick disease, Non-ketotic hyperglycinemia, Noonan syndrome, Noonan syndrome-like disorder, Norum disease, Macular degeneration, N-terminal acetyltransferase deficiency, Oculocutaneous albinism, Oculodentodigital dysplasia, Ohdo syndrome, Optic nerve aplasia, Ornithine carbamoyltransferase deficiency, Orofaciodigital syndrome, Osteogenesis imperfecta, Osteopetrosis, Ovarian dysgenesis, Pachyonychia, Palmoplantar keratoderma, nonepidermolytic, Papillon-Lef\xc3\xa8vre syndrome, Haim-Munk syndrome, Periodontitis, Peeling skin syndrome, Pendred syndrome, Peroxisomal fatty acyl-coa reductase 1 disorder, Peroxisome biogenesis disorder, Pfeiffer syndrome, Phenylketonuria, Phenylketonuria, Hyperphenylalaninemia, non-PKU, Pituitary hormone deficiency, Pityriasis rubra pilaris, Polyarteritis nodosa, Polycystic kidney disease, Polycystic lipomembranous osteodysplasia, Polymicrogyria, Pontocerebellar hypoplasia, Porokeratosis, Posterior column ataxia, Primary erythromelalgia, hyperoxaluria, Progressive familial intrahepatic cholestasis, Progressive pseudorheumatoid dysplasia, Propionic acidemia, Pseudohermaphroditism, Pseudohypoaldosteronism, Pseudoxanthoma elasticum-like disorder, Purine-nucleoside phosphorylase deficiency, Pyridoxal 5-phosphate-dependent epilepsy, Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia, skeletal dysplasia, Reticular dysgenesis, Retinitis pigmentosa, Usher syndrome, Retinoblastoma, Retinopathy, RRM2B-related mitochondrial disease, Rubinstein-Taybi syndrome, Schnyder crystalline corneal dystrophy, Sebaceous tumor, Severe congenital neutropenia, Severe myoclonic epilepsy in infancy, Severe X-linked myotubular myopathy, onychodysplasia, facial dysmorphism, hypotrichosis, Short-rib thoracic dysplasia, Sialic acid storage disease, Sialidosis, Sideroblastic anemia, Small fiber neuropathy, Smith-Magenis syndrome, Sorsby fundus dystrophy, Spastic ataxia, Spastic paraplegia, Spermatogenic failure, Spherocytosis, Sphingomyelin/cholesterol lipidosis, Spinocerebellar ataxia, Split-hand/foot malformation, Spondyloepimetaphyseal dysplasia, Platyspondylic lethal skeletal dysplasia, Squamous cell carcinoma of the head and neck, Stargardt disease, Sucrase-isomaltase deficiency, Sudden infant death syndrome, Supravalvar aortic stenosis, Surfactant metabolism dysfunction, Tangier disease, Tatton-Brown-rahman syndrome, Thoracic aortic aneurysms and aortic dissections, Thrombophilia, Thyroid hormone resistance, TNF receptor-associated periodic fever syndrome (TRAPS), Tooth agenesis, Torsades de pointes, Transposition of great arteries, Treacher Collins syndrome, Tuberous sclerosis syndrome, Tyrosinase-negative oculocutaneous albinism, Tyrosinase-positive oculocutaneous albinism, Tyrosinemia, UDPglucose-4-epimerase deficiency, Ullrich congenital muscular dystrophy, Bethlem myopathy Usher syndrome, UV-sensitive syndrome, Van der Woude syndrome, popliteal pterygium syndrome, Very long chain acyl-CoA dehydrogenase deficiency, Vesicoureteral reflux, Vitreoretinochoroidopathy, Von Hippel-Lindau syndrome, von Willebrand disease, Waardenburg syndrome, Warsaw breakage syndrome, WFS1-Related Disorders, Wilson disease, Xeroderma pigmentosum, X-linked agammaglobulinemia, X-linked hereditary motor and sensory neuropathy, X-linked severe combined immunodeficiency, and Zellweger syndrome.

The development of nucleobase editing advances both the scope and effectiveness of genome editing. The nucleobase editors described here offer researchers a choice of editing with virtually no indel formation (NBE2), or more efficient editing with a low frequency (here, typically ≤1%) of indel formation (NBE3). That the product of base editing is, by definition, no longer a substrate likely contributes to editing efficiency by preventing subsequent product transformation, which can hamper traditional Cas9 applications. By removing the reliance on double-stranded DNA cleavage and stochastic DNA repair processes that vary greatly by cell state and cell type, nucleobase editing has the potential to expand the type of genome modifications that can be cleanly installed, the efficiency of these modifications, and the type of cells that are amenable to editing. It is likely that recent engineered Cas9 variants^(69,70, 82) or delivery methods⁷¹ with improved DNA specificity, as well as Cas9 variants with altered PAM specificities,⁷² can be integrated into this strategy to provide additional nucleobase editors with improved DNA specificity or that can target an even wider range of disease-associated mutations. These findings also suggest that engineering additional fusions of dCas9 with enzymes that catalyze additional nucleobase transformations will increase the fraction of the possible DNA base changes that can be made through nucleobase editing. These results also suggest architectures for the fusion of other DNA-modifying enzymes, including methylases and demathylases, that mau enable additional types of programmable genome and epigenome base editing.

Materials and Methods

Cloning. DNA sequences of all constructs and primers used in this paper are listed in the Supplementary Sequences. Plasmids containing genes encoding NBE1, NBE2, and NBE3 will be available from Addgene. PCR was performed using VeraSeq ULtra DNA polymerase (Enzymatics), or Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). NBE plasmids were constructed using USER cloning (New England Biolabs). Deaminase genes were synthesized as gBlocks Gene Fragments (Integrated DNA Technologies), and Cas9 genes were obtained from previously reported plasmids.¹⁸ Deaminase and fusion genes were cloned into pCMV (mammalian codon-optimized) or pET28b (E. coli codon-optimized) backbones. sgRNA expression plasmids were constructed using site-directed mutagenesis. Briefly, the primers listed in the Supplementary Sequences were 5′ phosphorylated using T4 Polynucleotide Kinase (New England Biolabs) according to the manufacturer's instructions. Next, PCR was performed using Q5 Hot Start High-Fidelity Polymerase (New England Biolabs) with the phosphorylated primers and the plasmid pFYF1320 (EGFP sgRNA expression plasmid) as a template according to the manufacturer's instructions. PCR products were incubated with DpnI (20 U, New England Biolabs) at 37° C. for 1 h, purified on a QIAprep spin column (Qiagen), and ligated using QuickLigase (New England Biolabs) according to the manufacturer's instructions. DNA vector amplification was carried out using Machi competent cells (ThermoFisher Scientific).

In vitro deaminase assay on ssDNA. Sequences of all ssDNA substrates are listed in the Supplementary Sequences. All Cy3-labelled substrates were obtained from Integrated DNA Technologies (IDT). Deaminases were expressed in vitro using the TNT T7 Quick Coupled Transcription/Translation Kit (Promega) according to the manufacturer's instructions using 1 μg of plasmid. Following protein expression, 5 μL of lysate was combined with 35 μL of ssDNA (1.8 μM) and USER enzyme (1 unit) in CutSmart buffer (New England Biolabs) (50 mM potassium acetate, 29 mM Trisacetate, 10 mM magnesium acetate, 100 ug/mL BSA, pH 7.9) and incubated at 37° C. for 2 h. Cleaved U-containing substrates were resolved from full-length unmodified substrates on a 10% TBE-urea gel (Bio-Rad).

Expression and purification of His6-rAPOBEC1-linker-dCas9 fusions. E. Coli BL21 STAR (DE3)-competent cells (ThermoFisher Scientific) were transformed with plasmids encoding pET28b-His₆-rAPOBEC-linker-dCas9 with GGS, (GGS)₃, (SEQ ID NO: 596) XTEN, or (GGS)₇ (SEQ ID NO: 597) linkers. The resulting expression strains were grown overnight in Luria-Bertani (LB) broth containing 100 μg/mL of kanamycin at 37° C. The cells were diluted 1:100 into the same growth medium and grown at 37° C. to OD₆₀₀=˜0.6. The culture was cooled to 4° C. over a period of 2 h, and isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added at 0.5 mM to induce protein expression. After ˜16 h, the cells were collected by centrifugation at 4,000 g and resuspended in lysis buffer (50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 7.0, 1 M NaCl, 20% glycerol, 10 mM tris(2-carboxyethyl)phosphine (TCEP, Soltec Ventures)). The cells were lysed by sonication (20 s pulse-on, 20 s pulse-off for 8 min total at 6 W output) and the lysate supernatant was isolated following centrifugation at 25,00 g for 15 nm. The lysate was incubated with His-Pur nickel-nitriloacetic acid (nickel-NTA) resin (ThermoFisher Scientific) at 4° C. for 1 h to capture the His-tagged fusion protein. The resin was transferred to a column and washed with 40 mL of lysis buffer. The His-tagged fusion protein was eluted in lysis buffer supplemented with 285 mM imidazole, and concentrated by ultrafiltration (Amicon-Millipore, 100-kDa molecular weight cut-off) to 1 mL total volume. The protein was diluted to 20 mL in low-salt purification buffer containing 50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 7.0, 0.1 M NaCl, 20% glycerol, 10 mM TCEP and loaded onto SP Sepharose Fast Flow resin (GE Life Sciences). The resin was washed with 40 mL of this low-salt buffer, and the protein eluted with 5 mL of activity buffer containing 50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 7.0, 0.5 M NaCl, 20% glycerol, 10 mM TCEP. The eluted proteins were quantified on a SDSPAGE gel.

In vitro transcription of sgRNAs. Linear DNA fragments containing the T7 promoter followed by the 20-bp sgRNA target sequence were transcribed in vitro using the primers listed in the Supplementary Sequences with the TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific) according to the manufacturer's instructions. sgRNA products were purified using the MEGAclear Kit (ThermoFisher Scientific) according to the manufacturer's instructions and quantified by UV absorbance.

Preparation of Cy3-conjugated dsDNA substrates. Sequences of 80-nucleotide unlabeled strands are listed in the Supplementary Sequences and were ordered as PAGE-purified oligonucleotides from IDT. The 25-nt Cy3-labeled primer listed in the Supplementary Sequences is complementary to the 3′ end of each 80-nt substrate. This primer was ordered as an HPLC-purified oligonucleotide from IDT. To generate the Cy3-labeled dsDNA substrates, the 80-nt strands (5 μL of a 100 μM solution) were combined with the Cy3-labeled primer (5 μL of a 100 M solution) in NEBuffer 2 (38.25 μL of a 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9 solution, New England Biolabs) with dNTPs (0.75 μL of a 100 mM solution) and heated to 95° C. for 5 min, followed by a gradual cooling to 45° C. at a rate of 0.1° C./s. After this annealing period, Klenow exo⁻ (5 U, New England Biolabs) was added and the reaction was incubated at 37° C. for 1 h. The solution was diluted with Buffer PB (250 μL, Qiagen) and isopropanol (50 μL) and purified on a QIAprep spin column (Qiagen), eluting with 50 μL of Tris buffer.

Deaminase assay on dsDNA. The purified fusion protein (20 μL of 1.9 μM in activity buffer) was combined with 1 equivalent of appropriate sgRNA and incubated at ambient temperature for 5 min. The Cy3-labeled dsDNA substrate was added to final concentration of 125 nM and the resulting solution was incubated at 37° C. for 2 h. The dsDNA was separated from the fusion by the addition of Buffer PB (100 μL, Qiagen) and isopropanol (25 μL) and purified on a EconoSpin micro spin column (Epoch Life Science), eluting with 20 μL of CutSmart buffer (New England Biolabs). USER enzyme (1 U, New England Biolabs) was added to the purified, edited dsDNA and incubated at 37° C. for 1 h. The Cy3-labeled strand was fully denatured from its complement by combining 5 μL of the reaction solution with 15 μL of a DMSO-based loading buffer (5 mM Tris, 0.5 mM EDTA, 12.5% glycerol, 0.02% bromophenol blue, 0.02% xylene cyan, 80% DMSO). The full-length C-containing substrate was separated from any cleaved, U-containing edited substrates on a 10% TBE-urea gel (Bio-Rad) and imaged on a GE Amersham Typhoon imager.

Preparation of in vitro-edited dsDNA for high-throughput sequencing (HTS). The oligonucleotides listed in the Supplementary Sequences were obtained from IDT. Complementary sequences were combined (5 μL of a 100 μM solution) in Tris buffer and annealed by heating to 95° C. for 5 min, followed by a gradual cooling to 45° C. at a rate of 0.1° C./s to generate 60-bp dsDNA substrates. Purified fusion protein (20 μL of 1.9 μM in activity buffer) was combined with 1 equivalent of appropriate sgRNA and incubated at ambient temperature for 5 min. The 60-mer dsDNA substrate was added to final concentration of 125 nM and the resulting solution was incubated at 37° C. for 2 h. The dsDNA was separated from the fusion by the addition of Buffer PB (100 μL, Qiagen) and isopropanol (25 μL) and purified on a EconoSpin micro spin column (Epoch Life Science), eluting with 20 μL of Tris buffer. The resulting edited DNA (1 μL was used as a template) was amplified by PCR using the HTS primer pairs specified in the Supplementary Sequences and VeraSeq Ultra (Enzymatics) according to the manufacturer's instructions with 13 cycles of amplification. PCR reaction products were purified using RapidTips (Diffinity Genomics), and the purified DNA was amplified by PCR with primers containing sequencing adapters, purified, and sequenced on a MiSeq high-throughput DNA sequencer (Illumina) as previously described.⁷³

Cell culture. HEK293T (ATCC CRL-3216), U2OS (ATCC-HTB-96) and ST486 cells (ATCC) were maintained in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin/streptomycin (1×, Amresco), at 37° C. with 5% C02. HCC1954 cells (ATCC CRL-2338) were maintained in RPMI-1640 medium (ThermoFisher Scientific) supplemented as described above. Immortalized rat astrocytes containing the ApoE4 isoform of the APOE gene (Taconic Biosciences) were cultured in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) and 200 μg/mL Geneticin (ThermoFisher Scientific).

Transfections. HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) and transfected at approximately 85% confluency. Briefly, 750 ng of NBE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of Lipofectamine 2000 (ThermoFisher Scientific) per well according to the manufacturer's protocol. Astrocytes, U2OS, HCC1954, HEK293T and ST486 cells were transfected using appropriate AMAXA NUCLEOFECTOR™ II programs according to manufacturer's instructions. 40 ng of infrared RFP (Addgene plasmid 45457)⁷⁴ was added to the nucleofection solution to assess nucleofection efficiencies in these cell lines. For astrocytes, U2OS, and ST486 cells, nucleofection efficiencies were 25%, 74%, and 92%, respectively. For HCC1954 cells, nucleofection efficiency was <10%. Therefore, following trypsinization, the HCC1954 cells were filtered through a 40 micron strainer (Fisher Scientific), and the nucleofected HCC1954 cells were collected on a Beckman Coulter MoFlo XDP Cell Sorter using the iRFP signal (abs 643 nm, em 670 nm). The other cells were used without enrichment of nucleofected cells.

High-throughput DNA sequencing of genomic DNA samples. Transfected cells were harvested after 3 d and the genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer's instructions. On-target and off-target genomic regions of interest were amplified by PCR with flanking HTS primer pairs listed in the Supplementary Sequences. PCR amplification was carried out with Phusion high-fidelity DNA polymerase (ThermoFisher) according to the manufacturer's instructions using 5 ng of genomic DNA as a template. Cycle numbers were determined separately for each primer pair as to ensure the reaction was stopped in the linear range of amplification (30, 28, 28, 28, 32, and 32 cycles for EMX1, FANCF, HEK293 site 2, HEK293 site 3, HEK293 site 4, and RNF2 primers, respectively). PCR products were purified using RapidTips (Diffinity Genomics). Purified DNA was amplified by PCR with primers containing sequencing adaptors. The products were gel-purified and quantified using the QUANT-IT™ PicoGreen dsDNA Assay Kit (ThermoFisher) and KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced on an Illumina MiSeq as previously described.⁷³

Data analysis. Sequencing reads were automatically demultiplexed using MiSeq Reporter (Illumina), and individual FASTQ files were analyzed with a custom Matlab script provided in the Supplementary Notes. Each read was pairwise aligned to the appropriate reference sequence using the Smith-Waterman algorithm. Base calls with a Q-score below 31 were replaced with N's and were thus excluded in calculating nucleotide frequencies. This treatment yields an expected MiSeq base-calling error rate of approximately 1 in 1,000. Aligned sequences in which the read and reference sequence contained no gaps were stored in an alignment table from which base frequencies could be tabulated for each locus.

Indel frequencies were quantified with a custom Matlab script shown in the Supplementary Notes using previously described criteria⁷¹. Sequencing reads were scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels might occur. If no exact matches were located, the read was excluded from analysis. If the length of this indel window exactly matched the reference sequence the read was classified as not containing an indel. If the indel window was two or more bases longer or shorter than the reference sequence, then the sequencing read was classified as an insertion or deletion, respectively.

All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.

Supplementary Sequences

Primers used for generating sgRNA transfection plasmids. rev_sgRNA_plasmid was used in all cases. The pFYF1320 plasmid was used as template as noted in Materials and Methods section. SEQ ID NOs: 329-338 appear from top to bottom below, respectively.

rev_sgRNA_plasmid GGTGTTTCGTCCTTTCCACAAG fwd_p53_Y163C GCTTGCAGATGGCCATGGCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC fwd_p53_N239D TGTCACACATGTAGTTGTAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC fwd_AP0E4_C158R GAAGCGCCTGGCAGTGTACCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC fwd_EMX1 GAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC fwd_FANCF GGAATCCCTTCTGCAGCACCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC fwd_HEK293_2 GAACACAAAGCATAGACTGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC fwd_HEK293_3 GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC fwd_HEK293_4 GGCACTGCGGCTGGAGGTGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC fwd_RNF2 GTCATCTTAGTCATTACCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

Sequences of all ssDNA substrates used in in vitro deaminase assays. SEQ ID NOs: 339-341 appear from top to bottom below, respectively.

rAPOBEC1 substrate Cy3-ATTATTATTATTCCGCGGATTTATTTATTTATTTATTTATTT hAID/pmCDA1 substrate Cy3-ATTATTATTATTAGCTATTTATTTATTTATTTATTTATTT hAPOBEC3G substrate Cy3-ATTATTATTATTCCCGGATTTATTTATTTATTTATTTATTT

Primers used for generating PCR products to serve as substrates for T7 transcription of sgRNAs for gel-based deaminase assay. rev_gRNA_T7 was used in all cases. The pFYF1320 plasmid was used as template as noted in Materials and Methods section. SEQ ID NOs: 342-365 appear from top to bottom below, respectively.

rev_sgRNA_T7 AAAAAAAGCACCGACTCGGTG fwd_sgRNA_T7_dsDNA_2 TAATACGACTCACTATAGGCCGCGGATTTATTTATTTAAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_3 TAATACGACTCACTATAGGTCCGCGGATTTATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_4 TAATACGACTCACTATAGGTTCCGCGGATTTATTTATTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_5 TAATACGACTCACTATAGGATTCCGCGGATTTATTTATTGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_6 TAATACGACTCACTATAGGTATTCCGCGGATTTATTTATGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_7 TAATACGACTCACTATAGGTTATTCCGCGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_S TAATACGACTCACTATAGGATTATTCCGCGGATTTATTTGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_9 TAATACGACTCACTATAGGTATTATTCCGCGGATTTATTGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_10 TAATACGACTCACTATAGGATTATTATCCGGGGATTTATGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_11 TAATACGACTCACTATAGGTATTATATTCCGCGGATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_12 TAATACGACTCACTATAGGTTATTATATTCCGCGGATTTGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_13 TAATACGACTCACTATAGGATTATTATATTCCGCGGATTGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_14 TAATACGACTCACTATAGGTATTATTATATTCCGCGGATGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_15 TAATACGACTCACTATAGGATTATTATTATTACCGCGGAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_18 TAATACGACTCACTATAGGATTATTATTATTATTACCGCGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_noC TAATACGACTCACTATAGGATATTAATTTATTTATTTAAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_dsDNA_ TAATACGACTCACTATAGGGGAGGACGTGCGCGGCCGCCGTTTTAGAGCTAGAAATAGCA APOE4_C112R fwd_sgRNA_T7_dsDNA TAATACGACTCACTATAGGGAAGCGCCTGGCAGTGTACCGTTTTAGAGCTAGAAATAGCA APOE4_C158R fwd_sgRNA_T7_dsDNA TAATACGACTCACTATAGGCTGTGGCAGTGGCACCAGAAGTTTTAGAGCTAGAAATAGCA CTNNB1_T41A fwd_sgRNA_T7_dsDNA TAATACGACTCACTATAGGCCTCCCGGCCGGCGGTATCCGTTTTAGAGCTAGAAATAGCA HRAS_081R fwd_sgRNA_T7_dsDNA TAATACGACTCACTATAGGGCTTGCAGATGGCCATGGCGGTTTTAGAGCTAGAAATAGCA 53_Y163C fwd_sgRNA_T7_dsDNA TAATACGACTCACTATAGGACACATGCAGTTGTAGTGGAGTTTTAGAGCTAGAAATAGCA 53_Y236C fwd_sgRNA_T7_dsDNA TAATACGACTCACTATAGGTGTCACACATGTAGTTGTAGGTTTTAGAGCTAGAAATAGCA 53_N23SD

Sequences of 80-nucleotide unlabeled strands and Cy3-labeled universal primer used in gel-based dsDNA deaminase assays. SEQ ID NOs: 366-390 appear from top to bottom below, respectively.

Cy3-primer Cy3-GTAGGTAGTTAGGATGAATGGAAGGTTGGTA dsDNA_2 GTCCATGGATCCAGAGGTCATCCATTAAATAAATAAATCCGCGGGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_3 GTCCATGGATCCAGAGGTCATCCATAAATAAATAAATCCGCGGAAGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_4 GTCCATGGATCCAGAGGTCATCCATAATAAATAAATCCGCGGAAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsSNA_5 GTCCATGGATCCAGAGGTCATCCAAATAAATAAATCGGCGGAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_5 GTCCATGGATCCAGAGGTCATCCAATAAATAAATCCGCGGAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAG dsDNA_7 GTCCATGGATCCAGAGGTCATCCATAAATAAATCCGCGGAATAAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_8 GTCCATGGATCCAGAGGTCATCCAAAATAAATCCGCGGAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDMA_3 GTCCATGGATCCAGAGGTCATCCAAATAAATCCGCGGAATAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDMA_10 GTCCATGGATCCAGAGGTCATCCAATAAATCCGCGGATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_11 GTCCATGGATCCAGAGGTCATCCATAAATCCGCGGAATATAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_12 GTCCATGGATCCAGAGGTCATCCAAAATCCGCGGAATATAATAAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_13 GTCCATGGATCCAGAGGTCATCCAAATCCGCGGAATATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_14 GTCCATGGATCCAGAGGTCATCCAATCCGCGGAATATAATAATAGGCTATACCAACCTTCCATTCATCCTAACTACGTAC dsDNA_15 GTCCATGGATCCAGAGGTCATCCAATCCGCGGTAATAATAATAAGGCTATACCAACCTTCCATTCATCCTAACTACGTAC dsDNA_18 GTCCATGGATGCAGAGGTCATCCAGCGGTAATAATAATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_noC GTCCATGGATCCAGAGGTCATCCATTAAATAAATAAATTAATATTACTATACCAACCTTCCATTCATCCTAACTACCTAC dsDNA_8U 5Cy3-GTAGGTGTTAGGATGAATGGAAGGTTGGTAGATATTATCUGCGGATTTATTGGATGAGACCTCTGGATCCATGGACAT dsDNA_APOE_ GCACCTCGCCGCGGTACTGCACCAGGCGGCCGCGCACGTCCTCCATGTCTACCAACCTTCCATTCATCCTAACTACCTAC C112R d5DNA_APOE_ CGGCGCCCTCGCGGGCCCCGGCCTGGTACACTGCCAGGCGCTTCTGCAGTAOCAACCTTCCATTCATCCTAACTACCTAC C158R dsDNA_CTNN5 GTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTGCCACAGCTCCTTACCAACCTTCCATTCATCCTAACTACCTAC T41A dsDNA_HRAS_ GGAGACGTGCCTGTTGGACATCCTGGATAOCGCCGGaCCGGGAGGAGTACTACCAACCTTCCATTCATCCTAACTACCTAC Q61R dsDNA_p53 ACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTGCAAGCAGTCATACCAACCTTCCATTCATCCTAACTACCTAC Y163C dsDNA_p53_ AGGTTGGCTCTGACTGTACCACCATCCACTACAACTGCATGTGTAACAGTACCAACCTTCCATTCATCCTAACTACCTAC Y236C dsDNA_p53_ TGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTGACAGTTCCTACCAACCTTCCATTCATCCTAACTACCTAC N239D

Primers used for generating PCR products to serve as substrates for T7 transcription of sgRNAs for high-throughput sequencing. rev_gRNA_T7 (above) was used in all cases. The pFYF1320 plasmid was used as template as noted in Materials and Methods section. SEQ ID NOs: 391-442 appear from top to bottom below, respectively.

fwd_sgRNA_T7_HTS_base TAATACGACTCACTATAGGTTATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_1A TAATACGACTCACTATAGGATATTTCGTGGATTTATTTA3TTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_1C TAATACGACTCACTATAGGCTATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_1G TAATACGACTCACTATAGGGTATTTCGTGSATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_2A TAATACGACTCACTATAGGTAATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_2C TAATACGACTCACTATAGGTCATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_2G TAATACGACTCACTATAGGTGATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_3T TAATACGACTCACTATAGGTTTTTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_3C TAATACGACTCACTATAGGTTCTTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_3G TAATACGACTCACTATAGGTTGTTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_4A TAATACGACTCACTATAGGTTAATTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_4C TAATACGACTCACTATAGGTTACTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_4G TAATACGACTCACTATAGGTTAGTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_5A TAATACGACTCACTATAGGTTATATCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_5C TAATACGACTCACTATAGGTTATCTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_5G TAATACGACTCACTATAGGTTATGTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_6A TAATACGACTCACTATAGGTTATTACGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_5C TAATACGACTCACTATAGGTTATTCCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_6G TAATACGACTCACTATAGGTTATTGCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_8A TAATACGACTCACTATAGGTTATTTCATGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_8T TAATACGACTCACTATAGGTTATTTCTTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_8C TAATACGACTCACTATAGGTTATTTCCTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_9A TAATACGACTCACTATAGGTTATTTCGAGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_9C TAATACGACTCACTATAGGTTATTTCGCGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_9G TAATACGACTCACTATAGGTTATTTCGGGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_10A TAATACGACTCACTATAGGTTATTTCGTAGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_10T TAATACGACTCACTATAGGTTATTTCGTIGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_10C TAATACGACTCACTATAGGTTATTTCGTCGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_11A TAATACGACTCACTATAGGTTATTTCGTGAATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_11T TAATACGACTCACTATAGGTTATTTCGTGTATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_11C TAATACGACTCACTATAGGTTATTTCGTGCATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_12T TAATACGACTCACTATAGGTTATTTCGTGGTTTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_12C TAATACGACTCACTATAGGTTATTTCGTGGCTTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_12G TAATACGACTCACTATAGGTTATTTCGTGGGTTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_13A TAATACGACTCACTATAGGTTATTTCGTGGAATTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_13C TAATACGACTCACTATAGGTTATTTCGTGGACTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_13G TAATACGACTCACTATAGGTTATTTCGTGGAGTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_77_HTS_multiC TAATACGACTCACTATAGGTTCCCCCCCCGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_77_HTS_TCGCACCC_odd TAATACGACTCACTATAGGCGCACCCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_77_HTS_CCTCGCAC_odd TAATACGACTCACTATAGGCTCGCACGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_77_HTS_ACCCTCGC_odd TAATACGACTCACTATAGGCCCTCGCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_77_HTS_GCACCCTC_even TAATACGACTCACTATAGGCACCCTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_TCGCACCC_even TAATACGACTCACTATAGGTCGCACCCGTG3ATTTATTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_CCTCGCAC_even TAATACGACTCACTATAGGCCTCGCACGTGGATTTATTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_ACCCTCGC_even TAATACGACTCACTATAGGACCCTCGCGTG3ATTTATTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_GCACCCTC_even TAATACGACTCACTATAGGGCACCCTCGTGGATTTATTAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_EMX1 TAATACGACTCACTATAGGGAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_FANCF TAATACGACTCACTATAGGGGAATCCCTTCTGCAGCACCGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_HEK293_site2 TAATACGACTCACTATAGGGAACACAAAGCATAGACTGCGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_HEK293_site3 TAATACGACTCACTATAGGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_HEK293_siie4 TAATACGACTCACTATAGGGGCACTGCGGCTGGAGGTGGGTTTTAGAGCTAGAAATAGCA fwd_sgRNA_T7_HTS_RNF2 TAATACGACTCACTATAGGGTCATCTTAGTCATTACCTGGTTTTAGAGCTAGAAATAGCA

Sequences of in vitro-edited dsDNA for high-throughput sequencing (HTS). Shown are the sequences of edited strands. Reverse complements of all sequences shown were also obtained. dsDNA substrates were obtained by annealing complementary strands as described in Materials and Methods. Oligonucleotides representing the EMX1, FANCF, HEK293 site 2, HEK293 site 3, HEK293 site 4, and RNF2 loci were originally designed for use in the gel-based deaminase assay and therefore have the same 25-nt sequence on their 5′-ends (matching that of the Cy3-primer). SEQ ID NOs: 443-494 appear from top to bottom below, respectively.

Base sequence ACGTAAACGGCCACAAGTTCTTATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 1A ACGTAAACGGCCACAAGTTCATATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 1C ACGTAAACGGCCACAAGTTCCTATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 1G ACGTAAACGGCCACAAGTTCGTATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 2A ACGTAAACGGCCACAAGTTCTAATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 2C ACGTAAACGGCCACAAGTTCTCATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 2G ACGTAAACGGCCACAAGTTCTGATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 3T ACGTAAACGGCCACAAGTTCTTTTTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 3C ACGTAAACGGCCACAAGTTCTTCTTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 3G ACGTAAACGGCCACAAGTTCTTGTTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 4A ACGTAAACGGCCACAAGTTCTTAATTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 4C ACGTAAACGGCCACAAGTTCTTACTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 4G ACGTAAACGGCCACAAGTTCTTAGTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 5A ACGTAAACGGCCACAAGTTCTTATATCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 5C ACGTAAACGGCCACAAGTTCTTATCTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 5G ACGTAAACGGCCACAAGTTCTTATGTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 5A ACGTAAACGGCCACAAGTTCTTATTACGTGGATTTATTTATGGCATCTTCTTCAAGGACG 6C ACGTAAACGGCCACAAGTTCTTATTCCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 5G ACGTAAACGGCCACAAGTTCTTATTGCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 8A ACGTAAACGGCCACAAGTTCTTATTTCATGGATTTATTTATGGCATCTTCTTCAAGGACG 8T ACGTAAACGGCCACAAGTTCTTATTTCTTGGATTTATTTATGGCATCTTCTTCAAGGACG 8C ACGTAAACGGCCACAAGTTCTTATTTCCTGGATTTATTTATGGCATCTTCTTCAAGGACG 9A ACGTAAACGGCCACAAGTTCTTATTTCGAGGATTTATTTATGGCATCTTCTTCAAGGACG 9C ACGTAAACGGCCACAAGTTCTTATTTCGCGGATTTATTTATGGCATCTTCTTCAAGGACG 9G ACGTAAACGGCCACAAGTTCTTATTTCGGGGATTTATTTATGGCATCTTCTTCAAGGACG 10A ACGTAAACGGCCACAAGTTCTTATTTCGTAGATTTATTTATGGCATCTTCTTCAAGGACG 10T ACGTAAACGGCCACAAGTTCTTATTTCGTTGATTTATTTATGGCATCTTCTTCAAGGACG 10C ACGTAAACGGCCACAAGTTCTTATTTCGTCGATTTATTTATGGCATCTTCTTCAAGGACG 11A ACGTAAACGGCCACAAGTTCTTATTTCGTGAATTTATTTATGGCATCTTCTTCAAGGACG 11T ACGTAAACGGCCACAAGTTCTTATTTCGTGTATTTATTTATGGCATCTTCTTCAAGGACG 11C ACGTAAACGGCCACAAGTTCTTATTTCGTGCATTTATTTATGGCATCTTCTTCAAGGACG 12T ACGTAAACGGCCACAAGTTCTTATTTCGTGGTTTTATTTATGGCATCTTCTTCAAGGACG 12C ACGTAAACGGCCACAAGTTCTTATTTCGTGGCTTTATTTATGGCATCTTCTTCAAGGACG 12G ACGTAAACGGCCACAAGTTCTTATTTCGTGGGTTTATTTATGGCATCTTCTTCAAGGACG 13A ACGTAAACGGCCACAAGTTCTTATTTCGTGGAATTATTTATGGCATCTTCTTCAAGGACG 13C ACGTAAACGGCCACAAGTTCTTATTTCGTGGACTTATTTATGGCATCTTCTTCAAGGACG 13G ACGTAAACGGCCACAAGTTCTTATTTCGTGGAGTTATTTATGGCATCTTCTTCAAGGACG multiC ACGTAAACGGCCACAAGTTCTTCCCCCCCCGATTTATTTATGGCATCTTCTTCAAGGACG TCGCACCC_odd ACGTAAACGGCCACAAGTTTCGCACCCGTGGATTTATTTATGGCATCTTCTTCAAGGACG CCTCGCAC_odd ACGTAAACGGCCACAAGTTCCTCGCACGTGGATTTATTTATGGCATCTTCTTCAAGGACG ACCCTCGC_odd ACGTAAACGGCCACAAGTTACCCTCGCGTGGATTTATTTATGGCATCTTCTTCAAGGACG GCACCCTC_odd ACGTAAACGGCCACAAGTTGCACCCTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG TCGCACCC_even ACGTAAACGGCCACAAGTATTCGCACCCGTGGATTTATTATGGCATCTTCTTCAAGGACG CCTCGCAC_even ACGTAAACGGCCACAAGTATCCTCGCACGTGGATTTATTATGGCATCTTCTTCAAGGACG ACCCTCGC_even ACGTAAACGGCCACAAGTATACCCTCGCGTGGATTTATTATGGCATCTTCTTCAAGGACG GCACCCTC_even ACGTAAACGGCCACAAGTATGCACCCTCGTGGATTTATTATGGCATCTTCTTCAAGGACG EMX1_invitro GTAGGTAGTTAGGATGAATGGAAGGTTGGTAGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTG FANCF_invitro GTAGGTAGTTAGGATGAATGGAAGGTTGGTACTCATGGAATCCCTTCTGCAGCACCTGGATCGCTTTTCCGAGCTTCTGG HEK293_site2_ GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAACTGGAACACAAAGCATAGACTGCGGGGCGGGCCAGCCTGAATAGCTG invitro HEK293_site3_ GTAGGTAGTTAGGATGAATGGAAGGTTGGTACTTGGGGCCCAGACTGAGCACGTGATGGCAGAGGAAAGGAAGCCCTGCT invitro HEK293_site4_ GTAGGTAGTTAGGATGAATGGAAGGTTGGTACCGGTGGCACTGCGGCTGGAGGTGGGGGTTAAAGCGGAGACTCTGGTGC invitro RNF2_invitro GTAGGTAGTTAGGATGAATGGAAGGTTGGTATGGCAGTCATCTTAGTCATTACCTGAGGTCGTTGTAACTCATATAA

Primers for HTS of in vitro edited dsDNA. SEQ ID NOs: 495-503 appear from top to bottom below, respectively.

fwd_invitro_HTS ACACTCTTTCCCTACACGAGCTCTTCCGATCTNNNNACGTAAACGGCCACAA rev_invitro_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCGTCCTTGAAGAAGATGC fwd_invitro_HEK_targets ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTAGGTAGTTAGGATAATGGAA rev_EMX1_invitro TGGAGTTCAGACGTGTGCTCTTCCGATCTCACCGGTTGATGTGATGG rev_FANCF_invitro TGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGAAGCTCGGAAAAGC rev_HEK293_site2_invitro TGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCTATTCAGGCTGGC rev_HEK293_site3_invitro TGGAGTTCAGACGTGTGCTCTTCCGATCTAGCAGGGCTTCCTTTC rev_HEK293_site4_invitro TGGAGTTCAGACGTGTGCTCTTCCGATCTGCACCAGAGTCTCCG rev_RNF2_invitro TGGAGTTCAGACGTGTGCTCTTCCGATCTTTATATGAGTTACAACGAACACC

Primers for HTS of on-target and off-target sites from all mammalian cell culture experiments. SEQ ID NOs: 504-579 and 1869-1900 appear from top to bottom below, respectively.

fwd_EMX1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGCTCAGCCTGACTGTTGA rev_EMX1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGTGGGTTTGTGGTTGC fwd_FANCF_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCATTGCAGAGAGGCGTATCA rev_FANCF_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGTCCCAGGTGCTGAC fwd_HEK293_site2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGCCCCATCTGTCAAACT rev_HEK293_site2_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTGAATGGATTCCTTGGAAACAATGA fwd_HEK293_Site3_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATGTGGGCTGCCTAGAAAGG rev_HEK293_sit93_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCCCAGCCAAACTTGTCAACC fwd_HEK293_site4_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAACCCAGGTAGCCAGAGAC rev_HEK293_site4_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTTCAACCCGAACGGAG fwd_RNF2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTCTTCTTTATTTCCAGCAATGT rev_RNF2_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGTTTTCATGTTCTAAAAATGTATCCCA fwd_p53_Y163C_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTACAGTACTCCCCTGCCCTC rev_p53_Y163C_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTGCTCACCATCGCTATCT fwd_p53_N239D_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTCATCTTGGGCCTGTGTT rev_p53_N239D_HTS  TGGAGTTCAGACGTGTGCTCTTCCGATCTAAATCGGTAAGAGGTGGGCC fwd_APOE4_C15SR_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCGGACATGGAGGACGTG rev_AFOE4_C15SR_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTTCCACCAGGGGCCC fwd_EMX1_off1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCCAATCATTGATGCTTTT rev_EMX1_off1_HTS  TGGAGTTCAGACGTGTGCTCTTCCGATCTAGAAACATTTACCATAGACTATCACCT fwd_EMX1_off2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGTAGCCTCTTTCTCAATGTGC rev_EWX1_off2_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTTTCACAAGGATGCAGTCT fwd_EMX1_off3_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAGCTAGACTCCGAGGGGA rev_EMX1_off3_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTCGTCCTGCTCTCACTT fwd_EMX1_off4_HTS ACACTCTTTCCCTACACGACGCTCTTCC3ATCTNNNNAGAGGCTGAAGAGGAAGACCA rev_EMX1_off4_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGGCCCAGCTGTGCATTCTAT fwd_EMX1_off6_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAAGAGGGCCAAGTCCTG rev_EMX1_off6_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCGAGGAGTGACAGCC fwd_EMX1_off7_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACTCCACCTGATCTCGGGG rev_EMX1_off7_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCGAGGAGGGAGGGAGCAG fwd_EMX1_off8_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACCACAAATGCCCAAGAGAC rev_EMX1_off8_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGACACAGTCAAGGGCCGG fwd_EMX1_off9_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCCACCTTTGAGGAGGCAAA rev_EMX1_off9_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTTCCATCTGAGAAGAGAGTGGT fwd_EMX1_off10_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCATACCTTGGCCCTTCCT rev_EMX1_off10_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCCTAGGCCCACACCAG fwd_FANCF_off1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAACCCACTGAAGAAGCAGGG rev_FANCF_off1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGCTTAATCCGGCTCCAT fwd_FANCF_off2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCAGTGTTTCCATCCCGAA rev_FANCF_off2_HTS TGGAGTTCAGACGTGTGOTCTTCCGATCTCCTCTGACCTCCACAACTCT fwd_FANCF_off3_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTGGGTACAGTTCTGCGTGT rev_FANCF_off3_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTCACTCTGAGCATCGCCAAG fwd_FANCF_off4_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGTTTAGAGCCAGTGAACTAGAG rev_FANCF_off4_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGCAAGACAAAATCCTCTTTATACTTTG fwd_FANCF_off5_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGGAGGGGACGGCCTTAC rev_FANCF_off5_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTCTGGCGAACATGGC fwd_FANCF_off6_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTGGTTAAGAGCATGGGC rev_FANCF_off6_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGATTGAGTCCCCACAGCACA fwd_FANCF_off7_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGTGTTTCCCATCCCCAA rev_FANCF_off7_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTGACCTCCACAACTGGAAAAT fwd_FANCF_off8_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCTTCCAGACCCACCTGAAG rev_FANCF_off*_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTACCGAGGAAAATTGCTTGTCG fwd_HEK293_site2_off1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGTGGAGAGTGAGTAAGCCA rev_HEK293_site2_off1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTACGGTAGGATGATTTCAGGCA fwd_HEK293_site2_cff2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACAAAGCAGTGTAGCTCAGG rev_HEK293_site2_off2_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTGGTACTCGAGTGTTATTCAG fwd_HEK293_site3_off1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCCCTGTTGACCTGGAGAA rev_HEK293_site3_off1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGTACTTGCCCTGACCA fwd_HEK293_site3_off2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTGGTGTTGACAGGGAGCAA rev_HEK293_site3_off2_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAGATGTGGGCAGAAGGG fwd_HEK293_site3_off3_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGAGAGGGAACAGAAGGGCT rev_HEK293_site3_off3_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCAAAGGCCCAAGAACCT fwd_HEK2S3_site3_off4_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTAGCACTTTGGAAGGTCG rev_HEK293_site3_off4_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTCATCTTAATCTGCTCAGCC fwd_HEK293_site3_off5_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAAAGGAGCAGCTCTTCCTGG rev_HEK293_site3_off5_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTGCACCATCTCCCACAA fwd_HEK293_site4_off1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCATGGCTTCTGAGACTCA rev_HEK293_site4_off1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTCCCTTGCACTCCCTGTCTTT fwd_HEK293_site4_off2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTGGCAATGGAGGCATTGG rev_HEK293_site4_off2_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGAGGCTGCCCATGAGAG fwd_HEK293_site4_off3_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGTCTGAGGCTCGAATCCTG rev_HEK293_site4_off3_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTGGCCTCCATATCCCTG fwd_HEK293_site4_off4_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTCCACCAGAACTCAGCCC rev_HEK293_site4_off4_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCGGTTCCTCCACAACAC fwd_HEK293_site4_off5_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACGGGAAGGACAGGAGAAC rev_HEK293_site4_off5_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGCAGGGGAGGGATAAAGCAG fwd_HEK293_site4_off6_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCACGGGAGATGGCTTATGT rev_HEK293_site4_off6_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCACATCCTCACTGTGCCACT fwd_HEK293_site4_off7_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCAGTCTCGGCCCCTCA rev_HEK293_site4_off7_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCACTGTAAAGCTCTTGGG fwd_HEK293_site4_off8_HTS AGACTCTTTCCCTACAC3ACGCTCTTCCGATCTNNNNAGGGTAGAGGGACAGAGCTG rev_HEX293_site4_off8_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGGACCCCACATAGTCAGTGC fwd_HEK293_site4_off9_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCTGTCAGCCCTATCTCCATC rev_HEK293_site4_off9_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTGGGCAATTAGGACAGGGAC fwd_HEK293_site4_off10_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCAGCGGAGGAGGTAGATTG rev_HEK293_site4_off10_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCAGTACCTGGAGTCCCGA fwd_HEK2_ChIP_off1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGACAGGCTCAGGAAAGCTGT rev_HEK2_ChIP_off1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTACACAAGCCTTTCTCCAGGG fwd_HEK2_ChIP_off2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAATAGGGGGTGAGACTGGGG rev_HEK2_ChIP_off2_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTCAGACGAGACTTGAGG fwd_HEK2_ChIP_off3_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCCAGCAGGAAAGGAATCT rev_HEK2_ChIp_off3_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTGACTGCACCTGTAGCCATG fwd_HEK2_ChIP_off4_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCAAGGAAATCACCCTGCCC rev_HEK2_ChIP_off4_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTAACTTCCTTGGTGTGCAGCT fwd_HEK2_ChIP_off5_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATOGGCTCAGCTACGTCATG rev_HEK2_ChIP_off5_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTAATAGCAGTGTGGT6GGCAA fWd_HEK3_ChIP_off1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCGCACATCCCTTGTCTCTCT rev_HEK3_ChIP_off1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCTACTGGAGCACACCCCAAG fwd_HEK3_ChIP_off2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGGGTCACGTAGCTTTGGTC rev_HEK3_ChIP_off2_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTGGTGGCCATGTGCAACTAA fwd_HEK3_ChIP_off3_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTACTACGTGCCAGCTCAGG rev_HEK3_ChIP_off3_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTACCTCCCCTCCTCACTAACC fwd_HEK3_ChIP_off4_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCCTCAGCTCCATTTCCTGT rev_HEK3_ChIP_off4_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTAACCTTTATGGCACCAGGGG fwd_HEK3_ChIP_off5_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAGCTCAGCATTAGCAGGCT rev_HEK3_ChIP_off5_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTTTCCTGGCTTTCCGATTCCC fwd_HEK4_ChIp_off1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGCAATTGGAGGAGGAGCT rev_HEK4_ChIp_off1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCACCAGCTACAGGCAGAACA fwd_HEK4_ChIP_off3_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTACCCCCAACACAGATGG rev_HEK4_ChIP_off3_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCCACACAACTCAGGTCCTCC

Sequences of single-stranded oligonucleotide donor templates (ssODNs) used in HDR studies.

EMX1 sense (SEQ ID NO: 580) TCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCAGAAC CGGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTT TGAGCAGAAGGAAGGGCTCCCATCACATCAACCGCGGTGGCGCATTGCCA CGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGT EMX1 antisense (SEQ ID NO: 581) ACCCTAGTCATTGGAGGTGACATCGATGTCCTCCCCATTGGCCTGCTTCG TGGCAATGCGCCACCGGTTGATGTGATGGGAGCCCTTCTTCTTCTGCTCA AACTCAGGCCCTTCCTCCTCCAGCTTCTGCCGTTTGTACTTTGTCCTCCG GTTCTGGAACCACACCTTCACCTGGGCCAGGGAGGGAGGGGCACAGATGA HEK293 site 3 sense (SEQ ID NO: 582) CATGCAATTAGTCTATTTCTGCTGCAAGTAAGGATGCATTTGTAGGCTTG ATGCTTTTTTTCTGCTTCTCCAGCCCTGGCCTGGGTCAATCCTTGGGGCT TAGACTGAGCACGTGATGGCAGAGGAAAGGAAGCCCTGCTTCCTCCAGAG GGCGTCGCAGGACAGCTTTTCCTAGACAGGGGCTAGTATGTGCAGCTCCT HEK293 site 3 antisense (SEQ ID NO: 583) AGGAGCTGCACATACTAGCCCCTGTCTAGGAAAAGCTGTCCTGCGACGCC CTCTGGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCACGTGCTCAGTCTA AGCCCCAAGGATTGACCCAGGCCAGGGCTGGAGAAGCAGATAAAAAGCAT CAAGCCTACAAATGCATGCTTACTTGCAGCAGAAATAGACTAATTGCATG HEK site 4 sense (SEQ ID NO: 584) GGCTGACAAAGGCCGGGCTGGGTGGAAGGAAGGGAGGAAGGGCGAGGCAG AGGGTCCAAAGCAGGATGACAGGCAGGGGCACCGCGGCGCCCCGGTGGCA TTGCGGCTGGAGGTGGGGGTTAAAGCGGAGACTCTGGTGCTGTGTGACTA CAGTGGGGGCCGTGCCCTCTCTGAGCCCCCGCCTCCAGGCCTGTGTGTGT HEK site 4 antisense (SEQ ID NO: 585) ACACACACAGGCCTGGAGGCGGGGGCTCAGAGAGGGCAGGGCCOCCACTG TAGTCACACAGCACCAGAGTCTCCGCTTTAACCCCCACCTCCAGCCGCAA TGCCACCGGGGCGCCGCGGTGCCCCTGCCTGTCATCCTGCTTTGGACCCT CTGCCTCGCCCTTCCTCCCTTCCTTCCACCCAGCCCGGCCTTTGTCAGCC APOE4 sense (SEQ ID NO: 743) AGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCG TAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGT ACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGC GAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGT APOE4 antisense (SEQ ID NO: 744) ACAGTGGCGGCCCGCACGCGGCCCTGTTCCACCAGGGGCCCCAGGCGCTC GCGGATGGCGCTGAGGCCGCGCTCGGCGCCCTCGCGGGCCCCGGCCTGGT ACACTGCCAGGCACTTCTGCAGGTCATCGGCATCGCGGAGGAGCCGCTTA CGCAGCTTGCGCAGGTGGGAGGCGAGGCGCACCCGCAGCTCCTCGGTGCT p53 Y163C sense (SEQ ID NO: 745) ACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCT GTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGC CATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCT GCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTGAGCAGCTGGGGCTG p53 Y163C antisense (SEQ ID NO: 746) CAGCCCCAGCTGCTCACCATCGCTATCTGAGCAGCGCTCATGGTGGGGGC AGCGCCTCACAACCTCCGTCATGTGCTGTGACTGCTTGTAGATGGCCATG GCGCGGACGCGGGTGCCGGGCGGGGGTGTGGAATCAACCCACAGCTGCAC AGGGCAGGTCTTGGCCAGTTGGCAAAACATCTTGTTGAGGGCAGGGGAGT

Deaminase Gene gBlocks Gene Fragments

hAID (SEQ ID NO: 586) CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGGATAGCCTCTTGATG AATAGACGCAAGTTCCTGTATCAGTTTAAAAACGTGAGATGGGCAAAAGG CCGACGAGAGACATATCTGTGCTATGTCGTTAAGCGCAGAGATTCAGCCA CCAGTTTCTCTCTCGACTTCGGCTACCTGOGGAACAAGAATGGTTGCCAT GTTGAGCTCCTGTTCCTGAGGTATATCAGCGACTGGGATTTGGACCCAGG GCGGTGCTATAGGGTGACATGGTTTACCTCCTGGTCACCTTGTTATGACT GCGCGCGGCATGTTGCCGATTTTCTGAGAGGGAACCCTAACCTGTCTCTG AGGATCTTCACCGCGCGACTGTACTTCTGTGAGGACCGGAAAGCCGAACC CGAGGGACTGAGACGCCTCCACAGAGCGGGTGTGCAGATTGCCATAATGA CCTTTAAGGACTACTTCTACTGCTGGAACACCTTCGTCGAAAATCACGAG CGGACTTTCAAGGCTTGGGAAGGATTGCATGAAAACAGCGTCAGGCTTTC CAGGCAGCTTCGCCGCATTCTTCTCCCGTTGTACGAGGTTGATGACCTCA GAGATGCCTTTAGAACACTGGGACTGTAGGCGGCCGCTCGATTGGTTTGG TGTGGCTCTAA rAPOBEC1 (mammalian) (SEQ ID NO: 587) CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGAGCTCAGAGACTGGC CCAGTGGCTGTGGACCCCACATTGAGACGGCGGATCGAGCCCCATGAGTT TGAGGTATTCTTCGATCCGAGAGAGCTCCGCAAGGAGACCTGCCTGCTTT ACGAAATTAATTGGGGGGGCCGGCACTCCATTTGGCGACATACATCACAG AACACTAACAAGCACGTCGAAGTCAACTTCATCGAGAAGTTCACGACAGA AAGATATTTCTGTCCGAACACAAGGTGCAGCATTACCTGGTTTCTCAGCT GGAGCCCATGCGGCGAATGTAGTAGGGCCATCACTGAATTCCTGTCAAGG TATCCCCACGTCACTCTGTTTATTTACATCGCAAGGCTGTACCACCACGC TGACCCCCGCAATCGACAAGGCCTGCGGGATTTGATCTCTTCAGGTGTGA CTATCCAAATTATGACTGAGCAGGAGTCAGGATACTGCTGGAGAAACTTT GTGAATTATAGCCCGAGTAATGAAGCCCACTGGCCTAGGTATCCCCATCT GTGGGTACGACTGTACGTTCTTGAACTGTACTGCATCATACTGGGCCTGC CTCCTTGTCTCAACATTCTGAGAAGGAAGCAGCCACAGCTGACATTCTTT ACCATCGCTCTTCAGTCTTGTCATTACCAGCGACTGCCCCCACACATTCT CTGGGCCACCGGGTTGAAATGAGCGGCCGCTCGATTGGTTTGGTGTGGCT CTAA pmCDA1 (SEQ ID NO: 588) CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGACAGACGCTGAATAT GTTAGGATCCATGAAAAACTGGATATCTATACATTTAAGAAGCAGTTCTT CAATAACAAAAAGTCAGTATCTCACAGATGCTATGTCCTGTTCGAACTCA AGAGAAGAGGAGAAAGGCGGGCCTGTTTCTGGGGGTACGCGGTTAATAAA CCCCAGTCCGGGACCGAGAGGGGGATTCACGCCGAGATCTTTTCAATTAG GAAGGTTGAAGAGTATCTTCGCGACAATCCCGGTCAGTTCACAATTAACT GGTACAGCTCCTGGAGCCCTTGCGCTGATTGCGCCGAGAAAATACTCGAA TGGTACAACCAGGAGTTGAGAGGCAATGGCCACACTCTCAAGATTTGGGC TTGCAAGCTTTACTACGAGAAGAACGCGAGAAATCAGATTGGCTTGTGGA ACCTCAGGGACAACGGGGTCGGGTTGAATGTTATGGTGTCCGAACATTAC CAGTGCTGTAGAAAGATCTTCATTCAGTCCAGTCACAATCAGCTGAACGA GAACAGATGGCTGGAGAAAACACTGAAACGGGCAGAGAAAAGGCGCTCAG AGCTGAGTATCATGATCCAGGTCAAAATCTCTGCATACAACCAAAAGCCC GGCTGTATAAGCGGCCGCTCGATTGGTTTGGTGTGGCTCTAA haPOBEC3G (SEQ ID NO: 589) CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGGAGCTGAAGTATCAC CCTGAGATGCGGTTTTTCCACTGGTTTAGTAAGTGGCGCAAACTTCATCG GGATCAGGAGTATGAAGTGACCTGGTATATCTCTTGGTCTCCCTGCACAA AATGTACACGCGACATGGCCACATTTCTGGCCGAGGATCCAAAGGTGACG CTCACAATCTTTGTGGCCCGCCTGTATTATTTCTGGGACCCGGATTATCA GGAGGCACTTAGGTCATTGTGCCAAAAGCGCGACGGACCACGGGCGACTA TGAAAATCATGAATTATGACGAATTCCAGCATTGCTGGAGTAAGTTTGTG TACAGCCAGCGGGAGCTGTTCGAGCCCTGGAACAATCTTCCCAAGTACTA CATACTGCTTCACATTATGTTGGGGGAGATCCTTCGGCACTCTATGGATC CTCCTACCTTTACGTTTAACTTTAATAATGAGCCTTGGGTTCGCGGGCGC CATGAAACCTATTTGTGCTACGAGGTCGAGCGGATGCATAATGATACGTG GGTCCTGCTGAATCAGAGGAGGGGGTTTCTGTGTAACCAGGCTCCACATA AACATGGATTTCTCGAGGGGCGGCACGCCGAACTGTGTTTCCTTGATGTG ATACCTTTCTGGAAGCTCGACCTTGATCAAGATTACAGGGTGACGTGTTT CACCTCCTGGTCACCCTGCTTCAGTTGCGCCCAAGAGATGGCTAATTTAT CAGTAAGAACAAGCATGTGTCCCTCTGTATTTTTACAGCCAGAATTTATG ATGACCAGGGCCGGTGCCAGGAGGGGCTGCGGACACTCGCTGAGGCGGGC GCGAAGATCAGCATAATGACATACTCCGAATTCAAACACTGTTGGGACAC TTTTGTGGACCACCAGGGCTGCCCATTTCAGCCGTGGGATGGGCTCGACG AACATAGTCAGGATCTCTCAGGCCGGCTGOGAGCCATATTGCAGAACCAG GAGAATTAGGCGGCCGCTCGATTGGTTTGGTGTGGCTCTAA rAPOBEC1 (E. Coli) (SEQ ID NO: 590) GGCCGGGGATTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAC CATGGATGTCTTCTGAAACCGGTCCGGTTGCGGTTGACCCGACCCTGCGT CGTCGTATCGAACCGCACGAATTCGAAGTTTTCTTCGACCCGCGTGAACT GCGTAAAGAAACCTGCCTGCTGTACGAAATCAACTGGGGTGGTCGTCACT CTATCTGGCGTCACACCTCTCAGAACACCAACAAACACGTTGAAGTTAAC TTCATCGAAAAATTCACCACCGAACGTTACTTCTGCCCGAACACCCGTTG CTCTATCACCTGGTTCCTGTCTTGGTCTCCGTGCGGTGAATGCTCTCGTG CGATCACCGAATTCCTGTCTCGTTACCCGCACGTTACCCTGTTCATCTAC ATCGCGCGTCTGTACCACCACGCGGACCCGCGTAACCGTCAGGGTCTGCG TGACCTGATCTCTTCTGGTGTTACCATCCAGATCATGACCGAACAGGAAT CTGGTTACTGCTGGCGTAACTTCGTTAACTACTCTCCGTCTAACGAAGCT GCACTGGCCGCGTTACTCCGCACCTGTGGGTTCGTCTGTACGTTCTGGAA CTGTACTGCATCATCCTGGGTCTGCCGCCGTGCCTGAACATCCTGCGTCG TAAACAGCCGCAGCTGACCTTCTTCACCATCGCGCTGCAGTCTTGCCACT ACCAGCGTCTGCCGCCGCACATCCTGTGGGCGACCGGTCTGAAAGGTGGT AGTGGAGGGAGCGGCGGTTCAATGGATAAGAAATAC

Amino Acid Sequences of NBE1, NBE2, and NBE3.

NBE1 for E. Coli expression (His₆-rAPOBEC1-XTEN-dCas9) (SEQ ID NO: 591) MGSSHHHHHHMSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTN KHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGL RDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQ LTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSK KFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSA SMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS GKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQL LNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTINL GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSPKKKRKV NBE1 for Mammalian expression (rAPOBEC1-XTEN-dCas9-NLS) (SEQ ID NO: 592) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQ IMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSC HYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLIQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLF IQLVQTYNQLFEENPINASGVDAKAISARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKHKDKDFLDNEENEDILE DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA NRNFIAGLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAKKGILQPVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRMIAKSEQEIGKATAKYFFYSNIM NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQNIVKKTEVQTGGFSKESILPKRN SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSPKKKRKV

Alternative NBE1 for Mammalian expression with human APOBEC1 (hAPOBEC1- XTEN-dCas9-NLS) (SEQ ID NO: 5737) MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTN HVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFW HMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLY ALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWRGSETP GTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKK NGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAA KNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYH DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQK NSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD YDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGG FDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKD LIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNE QKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTL TNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSPKKK RKV NBE2 (rAPOBEC1-XTEN-dCas9-UGI-NLS) (SEQ ID NO: 593) MSSETGPVAVDPTLRRRIEPHEFEVFFDFRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQ IMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSC HYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLF IQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILE DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIG NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV NBE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS) (SEQ ID NO: 594) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQ IMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSC HYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYFTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLF IQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPVYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILE DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDENIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIG NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVQDSNGENKIKMLSGGSPKKKRKV pmCDA1-XTEN-dCas9-UGI (bacteria) (SEQ ID NO: 5742) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQS GTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTL KIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWL EKTLKRAEKRRSELSIMIQVKILHTTKSPAVSGSETPGTSESATPESDKKYSIGLAIGTNSV GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLA EDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLP NEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGDSGGSMTNLSDIIEKETGKQLVIQESILMLPEEVEEVI GNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML pmCDA1-XTEN-nCas9-UGI-NLS (mammalian construct) (SEQ ID NO: 5743) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQS GTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTL KIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWL EKTLKRAEKRRSELSIMIQVKILHTTKSPAVSGSETPGTSESATPESDKKYSIGLAIGTNSV GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLA EDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLP NEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIG NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKR KV huAPOBEC3G-XTEN-dCas9-UGI (bacteria) (SEQ ID NO: 5744) MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLE GRHAELCFLDVIPFWKLDLDDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIY DDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGR LRAILQSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDR FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED IQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ ELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSMTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLL TSDAPEYKPWALVIQDSNGENKIKML huAPOBEC3G-XTEN-nCas9-UGI-NLS (mammalian construct) (SEQ ID NO: 5745) MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLE GRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIY DDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGR LRAILQSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDR FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED IQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYS VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV huAPOBEC3G (D316R_D317R)-XTEN-nCas9-UGI-NLS (mammalian construct) (SEQ ID NO: 5746) MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLE GRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIY RRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGR LRAILQSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDR FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED IQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV

Base Calling Matlab Script

(SEQ ID NO: 595) WTnuc = GCGGACATGGAGGACGTGCGCGGCCGCCTGGTGCAGTACCGC GGCGAGGTGCAGGCCATGCTCGGCCAGA GCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGT AAGCGGCTCCTCCGCGATGCCGATGAC CTGCAGAAGCGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGA GCGCGGCCTCAGCGCCATCCGCGAGCG CCTGGGGCCCCTGGTGGAACA G′;

%cycle through fastq files for different samples files=dir(‘*.fastq’); for d=1:20 filename=files(d).name; %read fastq file [header,seqs,qscore]=fastqread(filename); seqsLength=length(seqs);    % number of sequences seqsFile= strrep(filename,‘.fastq’,‘’);    % trims off .fastq %create a directory with the same name as fastq file ifexist(seqsFile,‘dir’);  error(‘Directory already exists. Please rename or move it before moving on.’); end mkdir(seqsFile);    % make directory wtLength=length(WTnuc);     % length of wildtype sequence %% aligning back to the wildtype nucleotide sequence % % MN is a matrix of the nucleotide alignment window=1:wtLength; sBLength=length(seqs);   % number of sequences % counts number of skips nSkips = 0; ALN=repmat(‘’,[sBLengthwtLength]); % iterate through each sequencing read for i = 1:sBLength %If you only have forward read fastq files leave as is %If you have R1 foward and R2 is reverse fastq files uncomment the %next four lines of code and the subsequent end statement % ifmod(d,2)==0; %  reverse=seqrcomplement(seqs{i}); %  [score,alignment,start]= swalign(reverse,WTnuc,‘Alphabet’,‘NT’); % else [score,alignment,start]=swalign(seqs{i},WTnuc,‘Alphabet’,‘NT’); % end % length of the sequencing read len= length(alignment(3,:)); % if there is a gap in the alignment, skip = 1 and we will  % throw away the entire read skip = 0;  for j = 1:len if (alignment(3,j) == ‘−’|| alignment(1,j) == ‘−’) skip = 1;      break;  end  %in addition if the qscore for any given base in the read is     %below 31 the nucleotide is turned into an N (fastq qscores that are not letters)  ifisletter(qscore{i}(start(1)+j−1)) else  alignment(1,j) = ‘N’;   end  end  if skip == 0 && len>10  ALN(i, start(2):(start(2)+length(alignment)−1))=alignment(1,:);    end  end % with the alignment matrices we can simply tally up the occurrences of % each nucleotide at each column in the alignment these % tallies ignore bases annotated as N % due to low qscores TallyNTD=zeros(5,wtLength); for i=1:wtLength TallyNTD(:,i)=[sum(ALN(:,i)==‘A’),sum(ALN(:,i)==‘C’),sum(ALN(:,i)==‘G’), sum(ALN(:,i)==‘T’),sum(ALN(:,i)==‘N’)]; end % we then save these tally matrices in the respective folder for % further processing save(strcat(seqsFile,‘/TallyNTD’),‘TallyNTD’); dlmwrite(strcat(seqsFile,‘/TallyNTD.txt’),TallyNTD,‘precision’, ‘%.3f’,‘newline’,‘pc’); end

INDEL Detection Matlab Script

(SEQ ID NO: 595) WTnuc = ′GCGGACATGGAGGACGTGCGCGGCCGCCTGGTGCAGTACCG CGGCGAGGTGCAGGCCATGCTCGGCCAGA GCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGT AAGCGGCTCCTCCGCGATGCCGATGAC CTGCAGAAGCGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGA GCGCGGCCTCAGCGCCATCCGCGAGCG CCTGGGGCCCCTGGTGGAACA G′;

%cycle through fastq files for different samples files=dir(‘*.fastq’); %specify start and width of indel window as well as length of each flank indelstart=154; width=30; flank=10; for d=1:3 filename=files(d).name; %read fastq file [header,seqs,qscore]=fastqread(filename); seqsLength=length(seqs); % number of sequences seqsFile =strcat(strrep(filename,‘.fastq’,‘’),‘_INDELS’); %create a directory with the same name as fastq file+_INDELS ifexist(seqsFile,‘dir’);    error(‘Directory already exists. Please rename or move it before moving on.’); end mkdir(seqsFile); % make directory wtLength=length(WTnuc); % length of wildtype sequence sBLength = length(seqs); % number of sequences % initialize counters and cell arrays nSkips = 0; notINDEL=0; ins={}; dels={}; NumIns=0; NumDels=0; % iterate through each sequencing read for i = 1:sBLength    %search for 10BP sequences that should flank both sides of the “INDEL WINDOW”   windowstart=strfind(seqs{i},WTnuc(indelstart-flank:indelstart));     windowend=strfind(seqs{i},WTnuc(indelstart+width:indelstart+width+flank )); %if the flanks are found proceed iflength(windowstart)==1&&length(windowend)==1 %if the sequence length matches the INDEL window length save as  %not INDEL if windowend-windowstart==width+flank notINDEL=notINDEL+1;  %if the sequence is two or more bases longer than the INDEL  %window length save as an Insertion elseif windowend-windowstart>=width+flank+2 NumIns=NumIns+1;  ins{NumDels}=seqs{i};  %if the sequence is two or more bases shorter than the INDEL  %window length save as a Deletion elseif windowend-windowstart<=width+flank−2 NumDels=NumDels+1;  dels{NumDels}=seqs{i};  %keep track of skipped sequences that are either one base  %shorter or longer than the INDEL window width else  nSkips=nSkips+1;  end  %keep track of skipped sequences that do not possess matching flank  %sequences else  nSkips=nSkips+1;   end  end  fid=fopen(strcat(seqsFile,‘/summary.txt’),‘wt’);  fprintf(fid, ‘Skipped reads %i\n not INDEL %i\n Insertions %i\n Deletions %i\n’, [nSkips, notINDEL, NumIns, NumDels]); fclose(fid);  save(strcat(seqsFile,‘/nSkips’),‘nSkips’); save(strcat(seqsFile,‘/notINDEL’),‘notINDEL’);  save(strcat(seqsFile,‘/NumIns’),‘NumIns’); save(strcat(seqsFile,‘/NumDels’),‘NumDels’);  save(strcat(seqsFile,‘/dels’),‘dels’);  C = dels;  fid=fopen(strcat(seqsFile,‘/dels.txt’),‘wt’); fprintf(fid,‘“%s”\n’,C{:});  fclose(fid);  save(strcat(seqsFile,‘/ins’),‘ins’); C = ins;  fid=fopen(strcat(seqsFile,‘/ins.txt’), ‘wt’); fprintf(fid,‘“%s”\n’,C{:});  fclose(fid);  end

Example 5: Cas9 Variant Sequences

The disclosure provides Cas9 variants, for example Cas9 proteins from one or more organisms, which may comprise one or more mutations (e.g., to generate dCas9 or Cas9 nickase). In some embodiments, one or more of the amino acid residues, identified below by an asterisk, of a Cas9 protein may be mutated. In some embodiments, the D10 and/or H840 residues of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, are mutated. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to any amino acid residue, except for D. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to an A. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is an H. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to any amino acid residue, except for H. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to an A. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is a D.

A number of Cas9 sequences from various species were aligned to determine whether corresponding homologous amino acid residues of D10 and H840 of SEQ ID NO: 10 or SEQ ID NO: 11 can be identified in other Cas9 proteins, allowing the generation of Cas9 variants with corresponding mutations of the homologous amino acid residues. The alignment was carried out using the NCBI Constraint-based Multiple Alignment Tool (COBALT(accessible at st-va.ncbi.nlm.nih.gov/tools/cobalt), with the following parameters. Alignment parameters: Gap penalties −11, −1; End-Gap penalties −5, −1. CDD Parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on. Query Clustering Parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular.

An exemplary alignment of four Cas9 sequences is provided below. The Cas9 sequences in the alignment are: Sequence 1 (S1): SEQ ID NO: 11|WP_010922251|gi 499224711|type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus pyogenes]; Sequence 2 (S2): SEQ ID NO: 12|WP_039695303|gi 746743737|type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus gallolyticus]; Sequence 3 (S3): SEQ ID NO: 13|WP_045635197|gi 782887988|type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus mitis]; Sequence 4 (S4): SEQ ID NO: 14|5AXW_A|gi 924443546|Staphylococcus aureus Cas9. The HNH domain (bold and underlined) and the RuvC domain (boxed) are identified for each of the four sequences. Amino acid residues 10 and 840 in S1 and the homologous amino acids in the aligned sequences are identified with an asterisk following the respective amino acid residue.

S1 1 --MDKK- YSIGLD*IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI--GALLEDSG--ET AEATRLKRTARRRYT 73 S2 1 --MTKKN YSIGLD*IGTNSVGWAVITDDYKVPAKKMKVLGNTDKKYIKKNLL--GALLFDSG--ET AEATRLKRTARRRYT 74 S3 1 --M-KKG YSIGLD*IGTNSVGFAVITDDYKVPSKKMKVLGNTDKRFIKKNLI--GALLFDEG--TT AEARRLKRTARRRYT 73 S4 1 GSHMKRN YILGLD*IGITSVGYGII--DYET-----------------RDVIDAGVRLFKEANVEN NEGRRSKRGARRLKR 61 S1 74 RRKNRICYLQEIFSNEMAKVDDSFEHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL 153 S2 75 RRKNRLRYLQEIFANETAKVDESFFQRLDESFLTDDDKTEDSHPIFGNKAEEDAYHQKFPTIYHLRKHLADSSEKADLRL 154 S3 74 RRKNRLRYLQEIFSEEMSKVDSSFEHRLDDSFLIPEDKRESKYPIFATLTEEKEYHKQFPTIYHLRKQLADSKEKTDLRL 153 S4 62 RRRHRIQRVKKLL--------------FDYNLLTD--------------------HSELSGINPYEARVKGLSQKLSEEE 107 S1 154 IYLALAHNIKERGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEK 233 S2 155 VYLALAHNIKERGHFLIEGELNAENTDVQKIFADFVGVYNRTFDDSHLSEITVDVASILTEKISKSRRLENLIKYYPTEK 234 S3 154 TYLALAHNIKYRGHFLYEEAFDIKNNDIQKIFNEFISIYDNTFEGSSLSGQNAQVEAIFTDKISKSAKRERVLKLEPDEK 233 S4 108 FSAALLHLAKRRG----------------------VHNVNEVEEDT---------------------------------- 131 S1 234 KNGLFGNLIALSLGLTPNEKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEIT 313 S2 235 KNTLFGNLIALALGLQPNEKTNFKLSEDAKLQFSKDTYEEDLEELLGKIGDDYADLFTSAKNLYDAILLSGILTVDDNST 314 S3 234 STGLFSEFLKLIVGNQADFKKHFDLEDKAPLQFSKDTYDEDLENLLGQIGDDFTDLFVSAKKLYDAILLSGILTVTDPST 313 S4 132 -----GNELS------------------TKEQISRN-------------------------------------------- 144 S1 314 KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKM--DGTEELLV 391 S2 315 KAPLSASMIKRYVEHHEDLEKLKEFIKANKSELYHDIFKDKNKNGYAGYIENGVKQDEFYKYLKNILSKIKIDGSDYFLD 394 S3 314 KAPLSASMIERYENHQNDLAALKQFIKNNLPEKYDEVFSDQSKDGYAGYIDGKTTQETFYKYIKNLLSKF--EGTDYFLD 391 S4 145 ----SKALEEKYVAELQ-------------------------------------------------LERLKKDG------ 165 S1 392 KLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE 471 S2 395 KIEREDFLRKQRTFDNGSIPHQIHLQEMHAILRRQGDYYPFLKEKQDRIEKILTFRIPYYVGPLVRKDSRFAWAEYRSDE 474 S3 392 KIEREDFLRKQRTFDNGSIPHQIHLQEMNAILRRQGEYYPFLKDNKEKIEKILTFRIPYYVGPLARGNRDFAWLTRNSDE 471 S4 166 --EVRGSINRFKTSD--------YVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGP--GEGSPFGW------K 227 S1 472 TITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL 551 S2 475 KITPWNFDKVIDKEKSAEKFITRMTLNDLYLPEEKVLPKHSHVYETYAVYNELTKIKYVNEQGKE-SFFDSNMKQEIFDH 553 S3 472 AIRPWNFEEIVDKASSAEDFINKMTNYDLYLPEEKVLPKHSLLYETFAVYNELTKVKFIAEGLRDYQFLDSGQKKQIVNQ 551 S4 228 DIKEW---------------YEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEK---LEYYEKFQIIEN 289 S1 552 LEKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR---FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 628 S2 554 VFKENRKVTKEKLLNYLNKEFFEYRIKDLIGLDKENKSFNASLGTYHDLKKIL-DKAFLDDKVNEEVIEDIIKTLTLFED 632 S3 552 LEKENRKVTEKDIIHYLHN-VDGYDGIELKGIEKQ---FNASLSTYHDLLKIIKDKEENDDAKNEAILENIVHTLTIFED 627 S4 290 VFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEF---TNLKVYHDIKDITARKEII---ENAELLDQIAKILTIYQS 363 S1 629 REMIEERLKTYAHLFDDKVMKQLKR-RRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED 707 S2 633 KDMIHERLQKYSDIFTANQLKKLER-RHYTGWGRLSYKLINGIRNKENNKTILDYLIDDGSANRNFMQLINDDTLPFKQI 711 S3 628 REMIKQRLAQYDSLFDEKVIKALTR-RHYTGWGKLSAKLINGICDKQTGNTILDYLIDDGKINRNFMQLINDDGLSFKEI 706 S4 364 SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDE------LWHTNDNQIAIFNRLKLVP--------- 428 S1 708

781 S2 712

784 S3 707

779 S4 429

505 S1 782 KRIEEGIKELGSQIL-------KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD----YDVDH*IVPQSFLKDD 850 S2 785 KKLQNSLKELGSNILNEEKPSYIEDKVENSHLQNDQLFLYYIQNGKDMYTGDELDIDHLSD----YDIDH*IIPQAFIKDD 860 S3 780 KRIEDSLKILASGL---DSNILKENPTDNNQLQNDRLFLYYLQNGKDMYTGEALDINQLSS----YDIDH*IIPQAFIKDD 852 S4 506 ERIEEIIRTTGK---------------ENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDH*IIPRSVSFDN 570 S1 851

922 S2 861

932 S3 853

924 S4 571

650 S1 923

1002 S2 933

1012 S3 925

1004 S4 651

712 S1 1003

1077 S2 1013

1083 S3 1005

1081 S4 713

764 S1 1078

1149 S2 1084

1158 S3 1082

1156 S4 765

835 S1 1150 EKGKSKKLKSVKELLGITIMERSSFEKNPI-DFLEAKG-----YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG 1223 S2 1159 EKGKAKKLKTVKELVGISIMERSFFEENPV-EFLENKG-----YHNIREDKLIKLPKYSLFEFEGGRRRLLASASELQKG 1232 S3 1157 EKGKAKKLKTVKTLVGITIMEKAAFEENPI-TFLENKG-----YHNVRKENILCLPKYSLFELENGRRRLLASAKELQKG 1230 S4 836 DPQTYQKLK--------LIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKV 907 S1 1224 NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEITEQISEFSKRVILADANLDKVLSAYNKH------ 1297 S2 1233 NEMVLPGYLVELLYHAHRADNF-----NSTEYLNYVSEHKKEFEKVLSCVEDFANLYVDVEKNLSKIRAVADSM------ 1301 S3 1231 NEIVLPVYLTTLLYHSKNVHKL-----DEPGHLEYIQKHRNEFKDLLNLVSEFSQKYVLADANLEKIKSLYADN------ 1299 S4 908 VKLSLKPYRFD-VYLDNGVYKFV-----TVKNLDVIK--KENYYEVNSKAYEEAKKLKKISNQAEFIASFYNNDLIKING 979 S1 1298 RDKPIREQAENITHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSIT--------GLYETRI----DLSQL 1365 S2 1302 DNFSIEEISNSFINLLTLTALGAPADFNFLGEKIPRKRYTSTKECLNATLIHQSIT--------GLYETRI----DLSKL 1369 S3 1300 EQADIEILANSFINLLTFTALGAPAAFKFFGKDIDRKRYTTVSEILNATLIHQSIT--------GLYETWI----DLSKL 1367 S4 980 ELYRVIGVNNDLLNRIEVNMIDITYR-EYLENMNDKRPPRIIKTIASKT---QSIKKYSTDILGNLYEVKSKKHPQIIKK 1055 S1 1366 GGD 1368 S2 1370 GEE 1372 S3 1368 GED 1370 S4 1056 G-- 1056

The alignment demonstrates that amino acid sequences and amino acid residues that are homologous to a reference Cas9 amino acid sequence or amino acid residue can be identified across Cas9 sequence variants, including, but not limited to Cas9 sequences from different species, by identifying the amino acid sequence or residue that aligns with the reference sequence or the reference residue using alignment programs and algorithms known in the art. This disclosure provides Cas9 variants in which one or more of the amino acid residues identified by an asterisk in SEQ ID NOs: 11-14 (e.g., S1, S2, S3, and S4, respectively) are mutated as described herein. The residues D10 and H840 in Cas9 of SEQ ID NO: 10 that correspond to the residues identified in SEQ ID NOs: 11-14 by an asterisk are referred to herein as “homologous” or “corresponding” residues. Such homologous residues can be identified by sequence alignment, e.g., as described above, and by identifying the sequence or residue that aligns with the reference sequence or residue. Similarly, mutations in Cas9 sequences that correspond to mutations identified in SEQ ID NO: 10 herein, e.g., mutations of residues 10, and 840 in SEQ ID NO: 10, are referred to herein as “homologous” or “corresponding” mutations. For example, the mutations corresponding to the D10A mutation in SEQ ID NO: 10 or S1 (SEQ ID NO: 11) for the four aligned sequences above are D11A for S2, D10A for S3, and D13A for S4; the corresponding mutations for H840A in SEQ ID NO: 10 or S1 (SEQ ID NO: 11) are H850A for S2, H842A for S3, and H560A for S4.

A total of 250 Cas9 sequences (SEQ ID NOs: 11-260) from different species were aligned using the same algorithm and alignment parameters outlined above. Amino acid residues homologous to residues 10, and 840 of SEQ ID NO: 10 were identified in the same manner as outlined above. The alignments are provided below. The HNH domain (bold and underlined) and the RuvC domain (boxed) are identified for each of the four sequences. Single residues corresponding to amino acid residues 10, and 840 in SEQ ID NO: 10 are boxed in SEQ ID NO: 11 in the alignments, allowing for the identification of the corresponding amino acid residues in the aligned sequences.

Lengthy table referenced here US20220220462A1-20220714-T00001 Please refer to the end of the specification for access instructions.

EXAMPLE 6: Next Generation C to T Editors

Other familes of cytidine deaminases as alterantives to base etitor 3 (BE3) constructs were examined. The different C to T editors were developed to have a narrow or different editing window, alternate sequence specificity to expand targetable substrates, and to have higher activity.

Using the methods described in Example 4, the pmCDA1 (cytidine deaminase 1 from Petromyzon marinus) activity at the HeK-3 site is evaluated (FIG. 42). The pmCDA1-nCas9-UGI-NLS (nCas9 indicates the Cas9 nickase described herein) construct is active on some sites (e.g., the C bases on the complementary strand at position 9, 5, 4, and 3) that are not accessible with rAPOBEC1 (BE3).

The pmCDA1 activity at the HeK-2 site is given in FIG. 43. The pmCDA1-XTEN-nCas9-UGI-NLS construct is active on sites adjacent to “G,” while rAPOBEC1 analog (BE3 construct) has low activity on “C”s that are adjacent to “G”s, e.g., the C base at position 11 on the complementary strand.

The percent of total sequencing reads with target C converted to T (FIG. 44), C converted to A (FIG. 45), and C converted to G (FIG. 46) are shown for CDA and APOBEC1 (the BE3 construct).

The huAPOBEC3G activity at the HeK-2 site is shown in FIG. 47. Two constructs were used: huAPOBEC3G-XTEN-nCas9-UGI-NLS and huAPOBEC3G*(D316R_D317R)-XTEN-nCas9-UGI-NLS. The huAPOBEC3G-XTEN-nCas9-UGI-NLS construct has different sequence specificity than rAPOBEC1 (BE3), as shown in FIG. 47, the editing window appears narrow, as indicated by APOBEC3G's descreased activity at position 4 compared to APOBEC1. Mutations made in huAPOBEC3G (D316R and D317R) increased ssDNA binding and resulted in an observable effect on expanding the sites which were edited (compare APOBEC3G with APOBEC3G_RR in FIG. 47). Mutations were chosen based on APOBEC3G crystal structure, see: Holden et al., Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implication. Nature. (2008); 121-4, the entire contents of which are incorporated herein by reference.

EXAMPLE 7: pmCDA1/huAPOBEC3G/rAPOBEC1 work in E. coli

LacZ selection optimization for the A to I conversion was performed using a bacterial strain with lacZ encoded on the F plasmid. A critical glutamic acid residue was mutated (e.g., GAG to GGG, Glu to Gly mutation) so that G to A by a cytidine deaminase would restore lacZ activity (FIG. 48). Strain CC102 was selected for the selection assay. APOBEC1 and CDA constructs were used in a selection assay to optimize G to A conversion.

To evaluate the the effect of copy number of the plasmids encoding the deaminase constructs on lacZ reversion frequency, the CDA and APOBEC1 deaminases were cloned into 4 plasmids with different replication origins (hence different copy numbers). SC 101, CloDF3, RSF1030, and PUC (copy number: PUC>RSF1030>CloDF3>SC101) and placed under an inducible promoter. The plasmids were individually transformed into E. coli cells harboring F plasmid containing the mutated LacZ gene. The expression of the deaminases were induced and LacZ activity was detected for each construct (FIG. 49). As shown in FIG. 49, CDA exhibited significantly higher activity than APOBEC1 in all instances, regardless of the plasmid copy number the deaminases were cloned in. Further, In terms of the copy number, the deaminase activity was positively correlated with the copy number of the plasmid they are cloned in, i.e., PUC>CloDF3>SC101.

LacZ reversions were confirmed by sequencing of the genomic DNA at the lacZ locus. To obtain the genomic DNA containing the corrected LacZ gene, cells were grown media containg X-gal, where cells having LacZ activity form blue colonies. Blue colonies were selected and grown in minimial media containing lactose. The cells were spun down, washed, and re-plated on minimal media plates (lactose). The blue colony at the highest dilution was then selected, and its genomic DNA was sequenced at the lacZ locus (FIG. 50).

A chloramphenicol reversion assay was designed to test the activity of different cytidine deaminases (e.g., CDA, and APOBEC1). A plasmid harboring a mutant CAT1 gene which confers chloramphenicol resistance to bacteria is constructed with RSF1030 as the replication origin. The mutant CAT1 gene encodings a CAT1 protein that has a H195R (CAC to CGC) mutation, rendering the protein inactive (FIG. 51). Deamination of the C base-paired to the G base in the CGC codon would convert the codon back to a CAC codon. restoring the activity of the protein. As shown in FIG. 52, CDA outperforms rAPOBEC in E. coli in restoring the acitivty of the chloramphenicol reisitance gene. The minimum inhibitory concentration (MIC) of chlor in S1030 with the selection plasmid (pNMG_ch_5) was approximately 1 μg/mL. Both rAPOBEC-XTEN-dCas9-UGI and CDA-XTEN-dCas9-UGI induced DNA correction on the selection plasmid (FIG. 53).

Next, the huAPOBEC3G-XTEN-dCas9-UGI protein was tested in the same assay. Interestingly, huAPOBEC3G-XTEN-dCas9-UGI exhibited different sequence specificity than the rAPOBEC1-XTEN-dCas9-UGI fusion protein. Only position 8 was edited with APOBEC3G-XTEN-dCas9-UGI fusion, as compared to the rAPOBEC11-XTEN-dCas9-UGIfusion (in which positions 3, 6, and 8 were edited) (FIG. 54).

EXAMPLE 8: C to T Base Editors with Less Off Target Editing

Current base editing technologies allow for the sequence-specific conversion of a C:G base pair into a T:A base pair in genomic DNA. This is done via the direct catalytic conversion of cytosine to uracil by a cytidine deaminase enzyme and thus, unlike traditional genome editing technologies, does not introduce double-stranded DNA breaks (DSBs) into the DNA as a first step. See, Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., and Liu, D. R. (2016), “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533, 420-424; the entire contents of which are incorporated by reference herein. Instead, catalytically dead SpCas9 (dCas9) or a SpCas9 nickase (dCas9(A840H)) is tethered to a cytidine deaminase enzyme such as rAPOBEC 1, pmCDA 1, or hAPOBEC3G. The genomic locus of interest is encoded by an sgRNA, and DNA binding and local denaturation is facilitated by the dCas9 portion of the fusion. However, just as wt dCas9 and wt Cas9 exhibit off-target DNA binding and cleavage, current base editors also exhibit C to T editing at Cas9 off-target loci, which limits their therapeutic usefulness.

It has been reported that the introduction of just three to four mutations into SpCas9 that neutralize nonspecific electrostatic interactions between the protein and the sugar-phosphate backbone of its target DNA, increases the DNA binding specificity of SpCas9. See. Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., and Joung, J. K. (2016) “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495; and Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., and Zhang, F. (2015) “Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84-88: the entire contents of each are hereby incorporated by reference herein. Four reported neutralizing mutations were therefore incorporated into the initially reported base editor BE3 (SEQ ID NO: 285), and found that off-target C to T editing of this enzyme is also drastically reduced (FIG. 55 ), with no decrease in on-target editing (FIG. 56).

As shown in FIG. 55, HEK293T cells were transfected with plasmids expressing BE3 or HF-BE3 and a sgRNA matching the EMX1 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target locus, plus the top ten known Cas9 off-target loci for the EMX1 sgRNA, as previously determined by Joung and coworkers using the GUIDE-seq method. See Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015) “GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.” Nat Biotech 33, 187-197; the entire contents of which are incorporated by reference herein. EMX1 off-target 5 locus did not amplify and is not shown. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed (FIG. 55). Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE3 and HF-BE3.

In FIG. 56, HEK293T cells were transfected with plasmids expressing BE3 or HF-BE3 and sgRNAs matching the genomic loci indicated using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci. The percentage of total DNA sequencing reads with all four bases at the target Cs within each protospacer are shown for treatment with BE3 or HF-BE3 (FIG. 56). Frequencies of indel formation are shown as well.

Primary Protein Sequence  of HF-BE3 (SEQ ID NO: 285): MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEK VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY TGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY ETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS GGSPKKKRKV

EXAMPLE 9: Development of Base Editors that Use Cas9 Variants and Modulation of the Base Editor Processivity to Increase the Target Range and Precision of the Base Editing Technology

Unlike iraditional genome edicing platforms, base editing technology allows precise single nucleotide changes in the DNA without inducing double-stranded breaks(DSBs). See, Komor, A. C. et al. Nature 533, 420-424 (2016). The current generation of base editor uses the NGG PAM exclusively. This limits its ability to edit desired bases within the genome, as the base editor needs to be placed at a precise location where the target base is placed within a 4-base region (the ‘deamination window’), approximately 15 bases upstream of the PAM. See, Komor, A. C. et al. Nature 533, 420-424 (2016). Moreover, due to the high processivity of cytidine deaminase, the base editor may convert all cytidines within its deamination window into thymidines, which could induce amino acid changes other than the one desired by the researcher. See, Komor, A. C. et al. Nature 533, 420-424 (2016).

Expanding the Scope of Base Editing Through the Development of Base Editors with Cas9 Variants

Cas9 homologs and other RNA-guided DNA binders that have different PAM specificities were incorporated into the base editor architecture. See, Kleinstiver, B. P. et al. Nature 523, 481-485 (2015); Kleinstiver, B. P. et al. Nature Biotechnology 33, 1293-1298 (2015); and Zetsche, B. et al. Cell 163, 759-771 (2015); the entire contents of each are incorporated by reference herein. Furthermore, innovations that have broadened the PAM specificities of various Cas9 proteins were also incorporated to expand the target reach of the base editor even more. See, Kleinstiver, B. P. et al. Nature 523, 481-485 (2015); and Kleinstiver, B. P. et al. Nature Biotechnology 33, 1293-1298 (2015). The current palette of base editors is summarized in Table 4.

TABLE 4 New base editors made from Cas9 Variants Base Editor Reference for Species PAM Name Cas9 variant S. pyogenes ...NGG BE3 Wild-type ...NGA VQR BE3 or Kleinstiver, B. P. EQR BE3 et al. ...NGCG VRER BE3 Kleinstiver, B. P. et al. S. aureus ...NNGRRT SaBE3 Wild-type ...NNNRRT SaKKH BE3 Kleinstiver, B. P. et al. L. bacterium TTTN... dCpf1 BE2 Zetsche, B. et al.

Modulating Base Editor's Processivity Through Site-Directed Mutagenesis of rAPOBEC1

It was reasoned that the processivity of the base editor could be modulated by making point mutations in the deaminase enzyme. The incorporation of mutations that slightly reduce the catalytic activity of deaminase in which the base editor could still catalyze on average one round of cytidine deamination but was unlikely to access and catalyze another deamination within the relevant timcscale were pursued. In effect, the resulting base editor would have a narrower deamination window.

rAPOBEC1 mutations probed in this work are listed in Table 5. Some of the mutations resulted in slight apparent impairment of rAPOBEC1 catalysis, which manifested as preferential editing of one cytidine over another when multiple cytidines are found within the deamination window. Combining some of these mutations had an additive effect, allowing the base editor to discriminate substrate cytidines with higher stringency. Some of the double mutants and the triple mutant allowed selective editing of one cytidine among multiple cytidines that are right next to one another (FIG. 57).

TABLE 5 rAPOBEC1 Point Mutations Investigated rAPOBEC1 mutation Corresponding mutation studied in this work in APOBEC3G Reference H121R/H122R D315R/D316R Holden, L. G. et al. R126A R320A Chen, K-M. et al. R126E R320E Chen, K-M. et al. R118A R313A Chen, K-M. et al. W90A W285A Chen, K-M. et al. W90Y W285Y R312E R326E

Base Editor PAM Expansion and Processivity Modulation

The next generation of base editors were designed to expand editable cytidines in the genome by using other RNA-guided DNA binders (FIG. 58). Using a NGG PAM only allows for a single target within the “window” whereas the use of multiple different PAMs allows for Cas9 to be positioned anywhere to effect selective deamination. A variety of new base editors have been created from Cas9 variants (FIG. 59 and Table 4). Different PAM sites (NGA, FIG. 60; NGCG, FIG. 61; NNGRRT, FIG. 62; and NNHRRT, FIG.63) were explored. Selective deamination was successfully achieved through kinetic modulation of cytidine deaminase point mutagenesis (FIG. 65 and Table 5).

The effect of various mutations on the deamination window was then investigated in cell culture using spacers with multiple cytidines (FIGS. 66 and 67).

Further, the effect of various mutations on different genomic sites with limited numbers of cytidines was examined (FIGS. 68 to 71). It was found that approximately one cytidine will be edited within the deamination windown in the spacer, while the rest of the cytidines will be left intact. Overall, the preference for editing is as follows: C₆>C₅<<C₇≈C₄.

Base Editing Using Cpf1

Cpf1, a Cas9 homolog, can be obtained as AsCpf1, LbCpf1, or from any other species. Schematics of fusion constructs, including BE2 and BE3 equivalents, are shown in FIG. 73. The BE2 equivalent uses catalytically inactive Cpf2 enzyme (dCpf1) instead of Cas9, while the BE3 equivalent includes the Cpf1 mutant, which nicks the target strand. The bottom schematic depicts different fusion architectures to combine the two innovations illustrated above it (FIG. 73). The base editing results of HEK293T cell TTTN PAM sites using Cpf1 BE2 were examined with different spacers (FIGS. 64A to 64C). In some embodiments, Cpf1 may be used in place of a Cas9 domain in any of the base editors provided herein. In some embodiments, the Cpf1 is a protein that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% identical to SEQ ID NO 313.

Full Protein Sequence of Cpf1 (SEQ ID NO: 313): MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI NLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN AIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM PQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

EXAMPLE 10: Increased Fidelity of Base Editing

Examining the difference between plasmid delivery of BE3 and HF-BE3, it was found that the two edit on-target loci with comparable efficiency (FIGS. 74 and 75). However, HF-BE3 edited off-target loci much less than BE3, meaning that HF-BE3 has a much higher DNA specificity than BE3 (FIG. 76). Deaminase protein lipofection to HEK cells demonstrated that protein delivery of BE3 results in comparable on-target activity, but much better specificity, than plasmid DNA delivery of BE3. Using improved transfection procedures and better plasmids (n=2), the experiment used the following conditions: protein delivery was 125 nM Cas9:sgRNA complex, plasmid delivery was 750 ng BE3/HF-BE3 plasmid+250 ng sgRNA plasmid, and lipofection was with 1.5 μL of Lipofectamine 2000 per well. EMX-1 off target site 2 and FANCF off-target site 1 showed the most off-target editing with BE3, compared to all of the off-targets assayed (FIGS. 77 and 78), while HEK-3 showed no significant editing at off-targets for any of the delivery methods (FIG. 79). HEK-4 shows some C-to-G editing on at the on-target site, while its off-target sites 1, 3, and 4 showed the most off-target editing of all the assayed sites (FIG. 80).

Delivery of BE3 Protein via Micro-Injection to Zebrafish

TYR guide RNAs were tested in an in vitro assay for sgRNA activity (FIGS. 81 and 82). The % HTS reads show shows many C residues were converted to T residues during a 2 h incubation with purified BE3 protein and PCR of the resulting product. Experiments used an 80-mer synthetic DNA substate with the target deamination site in 60 bp of its genomic context. This is not the same as % edited DNA strands because only one strand was nicked, so the product is not amplified by PCR. The proportion of HTS reads edited is equal to x/(2-x), where x is the actual proportion of THS reads edited. For 60% editing, the actual proportion of bases edited is 75%. “Off target” is represents BE3 incubated with the same DNA substrate, while bound to an off-target sgRNA. It was found sgRNAs sgRH_13, sgHR_17, and possibly sgHR_ 16 appeared to be promising targets for in vivo injection experiments.

The delivery of BE3 protein in was tested in vivo in zebrafish. Zebrafish embryos (n=16-24) were injected with either scramled sgRNA, sgHR_13, sgHR_16, or sgHR_17 and purified BE3. Three embryos from each condition were analyzed independently (single embryo) and for each condition, all of the injected embryos were pooled and sequenced as a pool. The results are shown in FIGS. 83 to 85.

EXAMPLE 11: Uses of Base Editors to Treat Disease

Base editors or complexes provided herein (e.g., BE3) may be used to modify nucleic acids. For example, base editors may be used to change a cytosine to a thymine in a nucleic acid (e.g., DNA). Such changes may be made to, inter alia, alter the amino acid sequence of a protein, to destroy or create a start codon, to create a stop codon, to distupt splicing donors, to disrupt splicing acceptors or edit regulatory sequences. Examples of possible nucleotide changes are shown in FIG. 86.

Base editors or complexes provided herein (e.g., BE3) may be used to edit an isoform of Apolipoprotein E in a subject. For example, an Apolipoprotein E isoform may be edited to yield an isoform associated with a lower risk of developing Alzheimer's disease. Apolipoprotein E has four isoforms that differ at amino acids 112 and 158. APOE4 is the largest and most common genetic risk factor for late-onset Alzheimer's disease. Arginine residue 158 of APOE4, encoded by the nucleic acid sequence CGC, may be changed to a cysteine by using a base editor (e.g., BE3) to change the CGC nucleic acid sequence to TGC, which encodes cysteine at residue 158. This change yields an APOE3r isoform, which is associated with lower Alzheimer's disease risk. See FIG. 87.

It was tested whether base editor BE3 could be used to edit APOE4 to APOE3r in mouse astrocytes (FIG. 88). APOE 4 mouse astrocytes were nucleofected with Cas9+ template or BE3, targeting the nucleic acid encoding Arginine 158 of APOE4. The Cas9+ template yielded only 0.3% editing with 26% indels, while BE3 yielded 75% editing with 5% indels. Two additional base-edited cytosines are silent and do not yield changes to the amino acid sequence (FIG. 88).

Base editors or complexes provided herein may be used to treat prion protein diseases such as Creutzfeldt-Jakob disease and fatal familial insomnia, for example, by introducing mutations into a PRNP gene. Reverting PRNP mutations may not yield therapeutic results, and intels in PRNP may be pathogenic. Accordingly, it was tested whether PRNP could be mutated using base editors (e.g., BE3) to introduce a premature stop codon in the PRNP gene. BE3, associated with its guide RNA, was introduced into HEK cells or glioblastoma cells and was capable of editing the PRNP gene to change the encoded arginine at residue 37 to a stop codon. BE3 yielded 41% editing (FIG. 89).

Additional genes that may be edited include the following: APOE editing of Arg 112 and Arg 158 to treat increased Alzheimer's risk; APP editing of Ala 673 to decrease Alzheimer's risk; PRNP editing of Arg 37 to treat fatal familial insomnia and other prion protein diseases; DMD editing of the exons 23 and 51 splice sites to treat Duchenne muscular dystrophy; FTO editing of intron 1 to treat obesity risk; PDS editing of exon 8 to treat Pendred syndrome (genetic deafness); TMC1 editing of exon 8 to treat congenital hearing loss; CYBB editing of various patient-relevant mutations to treat chronic granulomatous disease. Additional diseases that may be treated using the base editors provided herein are shown in Table 6. below.

UGI also plays a key role. Knocking out UDG (which UGI inhibits) was shown to dramatically improve the cleanliness and efficiency of C to T base editing (FIG. 90). Furthermore, base editors with nickase and without UGI were shown to produce a mixture of outcomes, with very high indel rates (FIG. 91).

EXAMPLE 12: Expanding the Targeting Scope of Base Editing

Base editing is a new approach to genome editing that uses a fusion protein containing a catalylically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair to induce programmable, single-nucleotide C→T (or G→A) changes in DNA without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions¹. The development of five new C→T (or G→A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing by 2.5-fold are described herein. Additionally, new base editors containing mutated cytidine deaminase domains that narrow the width of the apparent editing window from approximately 5 nucleotides to 1 or 2 nucleotides were engineered, enabling the discrimination of neighboring C nucleotides that would previously be edited with comparable efficiency. Together, these developments substantially increase the targeting scope of base editing.

CRISPR-Cas9 nucleases have been widely used to mediate targeted genome editing². In most genome editing applications, Cas9 forms a complex with a single guide RNA (sgRNA) and induces a double-stranded DNA break (DSB) at the target site specified by the sgRNA sequence. Cells primarily respond to this DSB through the non-homologuous end-joining (NHEJ) repair pathway, which results in stochastic insertions or deletions (indels) that can cause frameshift mutations that disrupt the gene. In the presence of a donor DNA template with a high degree of homology to the sequences flanking the DSB, gene correction can be achieved through an alternative pathway known as homology directed repair (HDR).^(3,4) Unfortunately, under most non-perturbalive conditions HDR is inefficient, dependent on cell state and cell type, and dominated by a larger frequency of indels.^(3,4) As most of the known genetic variations associated with human disease are point mutations⁵, methods that can more efficiently and cleanly make precise point mutations are needed.

Base editing, which enables targeted replacement of a C:G base pair with a T:A base pair in a programmable manner without inducing DSBs¹. has been recently described. Base editing uses a fusion protein between a catalytically inactivated (dCas9) or nickase form of Streptococcus pyogenes Cas9 (SpCas9), a cytidine deaminase such as APOBEC1, and an inhibitor of base excision repair such as uracil glycosylase inhibitor (UGI) to convert cytidines into uridines within a five-nucleotide window specified by the sgRNA.¹ The third-generation base editor, BE3, converts C:G base pairs to T:A base pairs, including disease-relevant point mutations, in a variety of cell lines with higher efficiency and lower indel frequency than what can be achieved using other genome editing methods¹. Subsequent studies have validated the deaminase-dCas9 fusion approach in a variety of settings⁶⁻⁷.

Efficient editing by BE3 requires the presence of an NGG PAM that places the target C within a five-nucleotide window near the PAM-distal end of the protospacer (positions 4-8, counting the PAM as positions 21-23)¹. This PAM requirement substantially limits the number of sites in the human genome that can be efficiently targeted by BE3, as many sites of interest lack an NGG 13- to 17-nucleotides downstream of the target C. Moreover, the high activity and processivity of BE3 results in conversion of all Cs within the editing window to Ts, which can potentially introduce undesired changes to the target locus. Herein, new C:G to T:A base editors that address both of these limitations are described.

It was thought that any Cas9 homolog that binds DNA and forms an “R-loop” complex⁸ containing a single-stranded DNA bubble could in principle be converted into a base editor. These new base editors would expand the number of targetable loci by allowing non-NGG PAM sites to be edited. The Cas9 homolog from Staphylococcus aureus (SaCas9) is considerably smaller than SpCas9 (1053 vs. 1368 residues), can mediate efficient genome editing in mammalian cells, and requires an NNGRRT PAM⁹. SpCas9 was replaced with SaCas9 in BE3 to generate SaBE3 and transfected HEK293T cells with plasmids encoding SaBE3 and sgRNAs targeting six human genomic loci (FIGS. 92A and 92B). After 3 d, the genomic loci were subjected to high-throughput DNA sequencing (HTS) to quantify base editing efficiency. SaBE3 enabled C to T base editing of target Cs at a variety of genomic sites in human cells, with very high conversion efficiencies (approximately 50-75% of total DNA sequences converted from C to T, without enrichment for transfected cells) arising from targeting Cs at positions 6-11. The efficiency of SaBE3 on NNGRRT-containing target sites in general exceeded that of BE3 on NGG-containing target sites¹. Perhaps due to its higher average efficiency, SaBE3 can also result in detectable base editing at target Cs at positions outside of the canonical BE3 activity window (FIG. 92C). In comparison, BE3 showed significantly reduced editing under the same conditions (0-11%), in accordance with the known SpCas9 PAM preference (FIG. 106A)¹⁰. These data show that SaBE3 can facilitate very efficient base editing at sites not accessible to BE3.

The targeting range of base editors was further expanded by applying recently engineered Cas9 variants that expand or alter PAM specificities. Joung and coworkers recently reported three SpCas9 mutants that accept NGA (VQR-Cas9), NGAG (EQR-Cas9), or NGCG(VRER-Cas9) PAM sequences¹¹. In addition, Joung and coworkers engineered a SaCas9 variant containing three mutations (SaKKH-Cas9) that relax its PAM requirement to NNNRRT¹². The SpCas9 portion of BE3 was replaced with these four Cas9 variants to produce VQR-BE3, EQR-BE3, VRER-BE3, and SaKKH-BE3, which target NNNRRT, NGA, NGAG, and NGCG PAMs respectively. HEK293T cells were transfected with plasmids encoding these constructs and sgRNAs targeting six genomic loci for each new base editor, and measured C to T base conversions using HTS.

SaKKH-BE3 edited sites with NNNRRT PAMs with efficiencies up to 62% of treated, non-enriched cells (FIG. 92D). As expected, SaBE3 was unable to efficiently edit targets containing PAMs that were NNNHRRT (where H=A, C, or T) (FIG. 92D). VQR-BE3, EQR-BE3, and VRER-BE3 exhibited more modest, but still substantial base editing efficiencies of up to 50% of treated, non-enriched cells at genomic loci with the expected PAM requirements with an editing window similar to that of BE3 (FIGS. 92E and 92F). Base editing efficiencies of VQR-BE3, EQR-BE3, and VRER-BE3 in general closely paralleled the reported PAM requirements of the corresponding Cas9 nucleases; for example. EQR-BE3 was unable to efficiently edit targets containing NGAH PAM sequences (FIG. 92F). In contrast, BE3 was unable to edit sites with NGA or NGCG PAMs efficiently (0-3%), likely due to its PAM restrictions (FIG. 106B).

Collectively, the properties of SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, and VRER-BE3 establish that base editors exhibit a modularity that facilitates their ability to exploit Cas9 homologs and engineered variants.

Next, base editors with altered activity window widths were developed. All Cs within the activity window of BE3 can be efficiently converted to Ts¹. The ability to modulate the width of this window would be useful in cases in which it is important to edit only a subset of Cs present in the BE3 activity window.

The length of the linker between APOBEC1 and dCas9 was previously observed to modulate the number of bases that are accessible by APOBEC1 in vitro¹. In HEK293T cells, however, varying the linker length did not significantly modulate the width of the editing window, suggesting that in the complex cellular milieu, the relative orientation and flexibility of dCas9 and the cytidine deaminase are not strongly determined by linker length (FIG. 96). Next, it was thought that truncating the 540 end of the sgRNA might narrow the base editing window by reducing the length of single-stranded DNA accessible to the deaminase upon formation of the RNA-DNA heteroduplex. HEK293T cells were co-transfected with plasmids encoding BE3 and sgRNAs of different spacer lengths targeting a locus with multiple Cs in the editing window. No consistent changes in the width of base editing when using truncated sgRNAs with 17- to 19-base spacers were observed (FIGS. 95A to 95B). Truncating the sgRNA spacer to fewer than 17 bases resulted in large losses in activity (FIG. 95A).

As an alternative approach, it was thought that mutations to the deaminase domain might narrow the width of the editing window through multiple possible mechanisms. First, some mutations may alter substrate binding, the conformation of bound DNA, or substrate accessibility to the active site in ways that reduce tolerance for non-optimal presentation of a C to the deaminase active site. Second, because the high activity of APOBEC1 likely contributes to the deamination of multiple Cs per DNA binding event,^(1,13,14) mutations that reduce the catalytic efficiency of the deaminase domain of a base editor might prevent it from catalyzing successive rounds of deamination before dissociating from the DNA. Once any C:G to T:A editing event has taken place, the sgRNA no longer perfectly matches the target DNA sequence and re-binding of the base editor to the target locus should be less favorable. Both strategies were tested in an effort to discover new base editors that distinguish among multiple cytidines within the original editing window.

Given the absence of an available APOBEC1 structure, several mutations previously reported to modulate the catalytic activity of APOBEC3G, a cytidine deaminase from the same family that shares 42% sequence similarity of its active site-containing domain to that of APOBEC1, were identified¹⁵. Corresponding APOBEC1 mutations were incorporated into BE3 and evaluated their effect on base editing efficiency and editing window width in HEK293T cells at two C-rich genomic sites containing Cs at positions 3,4, 5, 6, 8, 9, 10, 12, 13, and 14 (site A); or containing Cs at positions 5, 6, 7, 8, 9, 10, 11, and 13 (site B).

The APOBEC1 mutations R118A and W90A each led to dramatic loss of base editing efficiency (FIG. 97C). R132E led to a general decrease in editing efficiency but did not change the substantially narrow the shape of the editing window (FIG. 97C). In contrast, several mutations that narrowed the width of the editing window while maintaining substantial editing efficiency were found (FIGS. 93A and 97C). The “editing window width” was defined to represent the artificially calculated window width within which editing efficiency exceeds the half-maximal value for that target. The editing window width of BE3 for the two C-rich genomic sites tested was 5.0 (site A) and 6.1 (site B) nucleotides.

R126 in APOBEC1 is predicted to interact with the phosphate backbone of ssDNA¹³. Previous studies have shown that introducing the corresponding mutation into APOBEC3G decreased catalysis by at least 5-fold¹⁴. Interestingly, when introduced into APOBEC1 in BE3, R126A and R126E increased or maintained activity relative to BE3 at the most strongly edited positions (C5, C6, and C7), while decreasing editing activity at other positions (FIGS. 93A and 97C). Each of these two mutations therefore narrowed the width of the editing window at site A and site B to 4.4 and 3.4 nucleotides (R126A), or to 4.2 and 3.1 nucleotides (R126E). respectively (FIGS. 93A and 97C).

W90 in APOBEC1 (corresponding to W285 in APOBEC3G) is predicted to form a hydrophobic pocket in the APOBEC3G active site and assist in substrate binding¹³. Mutating this residue to Ala abrogated APOBEC3G's catalytic activity¹³. In BE3, W90A almost completely abrogated base editing efficiency (FIG. 97C). In contrast, it was found that W90Y only modestly decreased base editing activity while narrowing the editing window width at site A and site B to 3.8 and 4.9 nucleotides, respectively (FIG. 93A). These results demonstrate that mutations to the cytidine deaminase domain can narrow the activity window width of the corresponding base editors.

W90Y, R126E, and R132E, the three mutations that narrowed the editing window without drastically reducing base editing activity, were combined into doubly and triply mulated base editors. The double mutant W90Y+R126E resulted in a base editor (YE1-BE3) with BE3-like maximal editing efficiencies, but substantially narrowed editing window width (width at site A and site B=2.9 and 3.0 nucleotides, respectively (FIG. 93A). The W90Y+R132E base editor (YE2-BE3) exhibited modestly lower editing efficiencies (averaging 1.4-fold lower maximal editing yields across the five sites tested compared with BE3), and also substantially narrowed editing window width (width at site A and site B=2.7 and 2.8 nucleotides, respectively) (FIG. 97C). The R126E+R132E double mutant (EE-BE3) showed similar maximal editing efficiencies and editing window width as YE2-BE3 (FIG. 97C). The triple mutant W90Y+R126E+R132E (YEE-BE3) exhibited 2.0-fold lower average maximal editing yields but very little editing beyond the C6 position and an editing window width of 2.1 and 1.4 nucleotides for site A and site B, respectively (FIG. 97C). These data taken together indicate that mutations in the cytidine deaminase domain can strongly affect editing window widths, in some cases with minimal or only modest effects on editing efficiency.

The base editing outcomes of BE3, YE 1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 were further compared in HEK293T cells targeting four well-studied human genomic sites that contain multiple Cs within the BE3 activity window¹. These target loci contained target Cs at positions 4 and 5 (HEK site 3), positions 4 and 6 (HEK site 2), positions 5 and 6 (EMX1), or positions 6, 7, 8, and 11 (FANCF). BE3 exhibited little (<1.2-fold) preference for editing any Cs within the position 4-8 activity window. In contrast, YE1-BE3, exhibited a 1.3-fold preference for editing C5 over C4 (HEK site 3), 2.6-fold preference for C6 over C4 (HEK site 2), 2.0-fold preference for C5 over C6 (EMX1), and 1.5-fold preference for C6 over C7 (FANCF) (FIG. 93B). YE2-BE3 and EE-BE3 exhibited somewhat greater positional specificity (narrower activity window) than YE1-BE3, averaging 2.4-fold preference for editing C5 over C4 (HEK site 3), 9.5-fold preference for C6 over C4 (HEK site 2), 2.9-fold preference for C5 over C6 (EMX1), and 2.6-fold preference for C7 over C6 (FANCF) (FIG. 93B). YEE-BE3 showed the greatest positional selectivity, with a 2.9-fold preference for editing C5 over C4 (HEK site 3), 29.7-fold preference for C6 over C4 (HEK site 2), 7.9-fold preference for C5 over C6 (EMX1), and 7.9-fold preference for C7 over C6 (FANCF) (FIG. 93B). The findings establish that mutant base editors can discriminate between adjacent Cs, even when both nucleotides are within the BE3 editing window.

The product distributions of these four mutants and BE3 were further analyzed by HTS to evaluate their apparent proccssivity. BE3 generated predominantly T4-T5 (HEK site 3), T4-T6 (HEK site 2), and T5-T6 (EMX1) products in treated HEK293T cells, resulting in, on average, 7.4-fold more products containing two Ts, than products containing a single T. In contrast, YE1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 showed substantially higher preferences for singly edited C4-T5, C4-T6, and T5-C6 products (FIG. 93C). YE1-BE3 yielded products with an average single-T to double-T product ratio of 1.4. YE2-BE3 and EE-BE3 yielded products with an average single-T to double-T product ratio of 4.3 and 5.1, respectively (FIG. 93C). Consistent with the above results, the YEE-BE3 triple mutant favored single-T products by an average of 14.3-fold across the three genomic loci. (FIG. 93C). For the target site in which only one C is within the target window (HEK site 4, at position C5), all four mutants exhibited comparable editing efficiencies as BE3 (FIG. 98). These findings indicate that these BE3 mutants have decreased apparent processivity and can favor the conversion of only a single C at target sites containing multiple Cs within the BE3 editing window. These data also suggest a positional preference of C5>C6>C7=C4 for these mutant base editors, although this preference could differ depending on the target sequence.

The window-modulating mutations in APOBEC1 were applied to VQR-BE3, allowing selective base editing of substrates at sites targeted by NGA PAM (FIG. 107A). However, when these mutations were applied to SaKKH-BE3, a linear decrease in base editing efficiency was observed without the improvement in substrate selectivity, suggesting a different kinetic equilibrium and substrate accessibility of this base editor than those of BE3 and its variants (FIG. 107B).

The five base editors with altered PAM specificities described in this study together increase the number of disease-associated mutations in the ClinVar database that can in principle be corrected by base editing by 2.5-fold (FIGS. 94A and 94B). Similarly, the development of base editors with narrowed editing windows approximately doubles the fraction of ClinVar entries with a properly positioned NGG PAM that can be corrected by base editing without comparable modification of a non-target C (from 31% for BE3 to 59% for YEE-BE3) (FIGS. 94A and 94B).

In summary, the targeting scope of base editing was substantially expanded by developing base editors that use Cas9 variants with different PAM specificities, and by developing a collection of deaminase mutants with varying editing window widths. In theory, base editing should be possible using other programmable DNA-binding proteins (such as Cpf1¹⁶) that create a bubble of single-stranded DNA that can serve as a substrate for a single-strand-specific nucleotide deaminase enzyme.

Materials and Methods

Cloning. PCR was performed using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). Plasmids for BE and sgRNA were constructed using USER cloning (New England Biolabs), obtained from previously reported plasmids¹. DNA vector amplification was carried out using NEB 10beta competent cells (New England Biolabs).

Cell culture. HEK293T (ATCC CRL-3216) were cultured in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS), at 37° C. with 5% CO₂. Immortalized rat astrocytes containing the ApoE4 isoform of the APOE gene (Taconic Biosciences) were maintained in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) and 200 μg/mL Geneticin (ThermoFisher Scientific).

Transfections. HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) and transfected at approximately 85% confluency. 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of Lipofectamine 2000 (ThermoFisher Scientific) per well according to the manufacturer's protocol.

High-throughput DNA sequencing of genomic DNA samples. Transfected cells were harvested after 3 d and the genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer's instructions. Genomic regions of interest were amplified by PCR with flanking HTS primer pairs listed in the Supplementary Sequences. PCR amplification was carried out with Phusion hot-start II DNA polymerase (ThermoFisher) according to the manufacturer's instructions. PCR products were purified using RapidTips (Diffinity Genomics). Secondary PCR was performed to attach sequencing adaptors. The products were gel-purified and quantified using the KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced on an Illumina MiSeq as previously described¹.

Data analysis. Nucleotide frequencies were assessed using a previously described MATLAB script¹. Briefly, the reads were aligned to the reference sequence via the Smith-Waterman algorithm. Base calls with Q-scores below 30 were replaced with a placeholder nucleotide (N). This quality threshold results in nucleotide frequencies with an expected theoretical error rate of 1 in 1000.

Analyses of base editing processivity were performed using a custom python script. This program trims sequencing reads to the 20 nucleotide protospacer sequence as determined by a perfect match for the 7 nucleotide sequences that should flank the target site. These targets were then consolidated and sorted by abundance to assess the frequency of base editing products.

Bioinformatic analysis of the ClinVar database of human disease-associated mutations was performed in a manner similar to that previously described but with small adjustments¹. These adjustments enable the identification of targets with PAMs of customizable length and sequence. In addition, this improved script includes a priority ranking of target C positions (C5>C6>C7>C8≈C4), thus enabling the identification of target sites in which the on-target C is cither the only cytosine within the window or is placed at a position with higher predicted editing efficiency than any off-target C within the editing window.

References for Example 12

-   1 Komor, A. C. et al. Programmable editing of a target base in     genomic DNA without double-stranded DNA cleavage. Nature 533,     420-424 (2016). -   2 Sander, J. D. & Joung. J. K. CRISPR-Cas systems for editing,     regulating and targeting genomes. Nature biotechnology 32, 347-355     (2014). -   3 Cong, L. et al. Multiplex genome engineering using CRISPR/Cas     systems. Science 339, 819-823(2013). -   4 Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system.     Nat. Protocols 8, 2281-2308 (2013). -   5 Landrum, M. J. et al. ClinVar: public archive of interpretations     of clinically relevant variants. Nucleic Acids Res. 44, D862-D868     (2015). -   6 Nishida, K. et al. Targeted nucleotide editing using hybrid     prokaryotic and vertebrate adaptive immune systems. Science 353,     aaf8729-1-8 (2016). -   7 Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables     efficient genomic diversification in mammalian cells. Nat. Methods     doi:10.1038/nmeth.4027 (2016). -   8 Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed     for DNA cleavage. Science 351, 867-71 (2016). -   9 Ran, F. A. et al. In vivo genome editing using Staphylococcus     aureus Cas9. Nature 520, 186-191 (2015). -   10 Zhang, Y. et al. Comparison of non-canonical PAMs for     CRISPR/Cas9-mediated DNA cleavage in human cells. Sci. Rep. 4,     (2014). -   11 Kleinsliver, B. P. et. al. Engineered CRISPR-Cas9 nucleases with     altered PAM specificities. Nature 523, 481-485 (2015). -   12 Kleinsliver, B. P. et. al. Broadening the targeting range of     Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat.     Biotechnol. 33, 1293-1298 (2015). -   13 Holden, L. G. et al. Crystal structure of the anti-viral APOBEC3G     catalytic domain and functional implications. Nature 452, 121-124     (2008). -   14 Chen, K.-M. et al. Structure of the DNA deaminase domain of the     HIV-1 restriction factor APOBEC3G. Nature 452, 116-119 (2008). -   15 Harris, R. S., Petersen-Mahrt, S. K. & Neuberger, M. S. RNA     Editing Enzyme APOBEC1 and Some of Its Homologs Can Act as DNA     Mutators. Molecular Cell 10, 1247-1253(2002). -   16 Zetsche, B. et al. Cpf1 Is a Single RNA-Guided Endonuclease of a     Class 2 CRISPR-Cas System. Cell 163, 759-771 (2015).

EXAMPLE 13

Using improved transfection procedures and better plasmids, biological replicates (n=3) were used to install the four HF mutations into the Cas9 portion of BE3. The muations do not significantly effect on-targeting editing with plasmid delivery (FIG. 99). At the tested concentration, BE3 protein delivery works; however, the on-target editing is lower than for plasmid delivery (FIG. 100). Protein delivery of BE3 with the HF mutations installed reduces on-targeting editing efficiency but still yields some edited cells (FIG. 101).

Both lipofection and installing HF mutations were shown to decrease off-target deamination events. For the four sites shown in FIG. 102, the off-target sitest (OT) with the highest GUIDE-Seq reads and deamination events were assayed (Komor el al., Nature, 2016). The specificity ratio was calculated by dividing the off-target editing by the on-target editing at the closest corresponding C. In cases where off-target editing was not detectable, the ratio was set to 100. Thus, a higher specificity ratio indicates a more specific construct. BE3 plasmid delivery showed much higher off-target/on-target editing than protein delivery of BE3, plasmid delivery of HF-BE3, or protein delivery of HF-BE3 (FIGS. 102 and 105).

Purified proteins HF-BE3 and BE3 were analyzed in vitro for their capabilities to convert C to T residues at different positions in the spacer with the most permissive motif. Both BE3 and HF-BE3 proteins were found to have the same “window” for base editing (FIGS. 103 and 104).

A list of the disease targets is given in Table 9. The base to be edited in Table 9 is indicated in bold and underlined.

TABLE 9 Base Editor Disease Targets GENE DISEASE SPACER PAM EDITOR DEFECT CELL RB1 RETINOBLASTOMA AAT C TAGTAAA AAAAGT SAKKH-BE3 SPLICING J82 TAAATTGATGT IMPAIRMENT PTEN CANCER GACCAA C GGCT TGA VQR-BE3 W111R MC116 AAGTGAAGA PIK3CA CANCER TC C TTTCTTCA ACTGGT SAKKH-BE3 K111R CRL-5853 CGGTTGCCT PIK3CA CANCER CTC C TGCTCAG AGA VQR-BE3 Q546R CRL-2505 TGATTTCAG TP53 CANCER TGT C ACACATG TGG YEE-BE3 N239D SNU475 TAGTTGTAG HRAS CANCER CCTCC C GGCCG AGG YEE-BE3 Q61R MC/CAR GCGGTATCC

TABLE 6 Exemplary diseases that may be treated using base editors.  gene Base Disease target symbol changed sgRNA (PAM) Base editor Prion disease PRNP R37* GGCAGCCGATACCCGGGGCA(GGG) BE3 GGGCAGCCGATACCCGGGGC(AGG) Pendred syndrome Slc26a4 c.919-2A>G TTATTGTCCGAAATAAAAGA(AGA) BE3 ATTGTCCGAAATAAAAGAAG(AGG) (VQR SaCas9) TTGTCCGAAATAAAAGAAGA(GGA) GTCCGAAATAAAAGAAGAGGAAAA(AAT) GTCCGAAATAAAAGAAGAGGAAAAA(ATT) Congenital  Tmc1 c.545A>G CAGGAAGCACGAGGCCACTG(AGG) BE3 deafness AACAGGAAGCACGAGGCCAC(TGA) YE-BE3 AGGAAGCACGAGGCCACTGA(GGA) YEE-BE3 Acquired  SNHL S33F TTGGATTCTGGAATCCATTC(TGG) BE3 deafness Alzheimer's  APP A673T TCTGCATCCATCTTCACTTC(AGA) BE3 VQR Disease Niemann-Pick  NPC1 I1061T CTTACAGCCAGTAATGTCAC(CGA) BE3 VQR Disease Type C The protospacer and PAM sequences are shown in the sgRNA (PAM) column. The PAM sequence is shown in parentheses and with the base to be edited indicated by underlining.

Lengthy table referenced here US20220220462A1-20220714-T00002 Please refer to the end of the specification for access instructions.

Lengthy table referenced here US20220220462A1-20220714-T00003 Please refer to the end of the specification for access instructions.

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EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220220462A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

1-302. (canceled)
 303. A complex comprising: (i) a Cas9 protein; (ii) a cytidine deaminase; and (iii) a uracil glycosylase inhibitor (UGI) protein; wherein the Cas9 protein, the cytidine deaminase, the UGI protein, and a guide RNA (gRNA) associate in a complex, and wherein the complex comprising the Cas9 protein, the cytidine deaminase, the UGI protein, and the gRNA deaminates a cytidine in the target nucleic acid sequence.
 304. The complex of claim 303, wherein the Cas9 protein is a Cas9 nickase (nCas9).
 305. The complex of claim 304, wherein the nCas9 comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence provided in SEQ ID NO: 10, and wherein the amino acid sequence provided in SEQ ID NO: 10 further comprises a D10A mutation, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, 4273, 4275, and
 4276. 306. The complex of claim 304, wherein the nCas9 comprises one or more of N497A, R661A, Q695A, or Q926A mutations in the amino acid sequence provided in SEQ ID NO: 10, or one or more corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260, 4273, 4275, and
 4276. 307. The complex of claim 303, wherein the Cas9 protein is a nuclease-inactive Cas9 (dCas9).
 308. The complex of claim 307, wherein the dCas9 comprises at least 85% sequence identity to the amino acid sequence provided in SEQ ID NO: 10, and wherein the amino acid sequence provided in SEQ ID NO: 10 further comprises a D10A mutation, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260.
 309. The complex of claim 308, wherein the dCas9 comprises an H840A mutation in the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260.
 310. The complex of claim 307, wherein the dCas9 comprises one or more of N497A, R661A, Q695A, or Q926A mutations in the amino acid sequence provided in SEQ ID NO: 10, or one or more corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260.
 311. The complex of claim 303, wherein the Cas9 protein is selected from the group consisting of a Staphylococcus aureus Cas9 protein (SaCas9), a nuclease inactive SaCas9 (dSaCas9), a SaCas9 nickase (nSaCas9), a Streptococcus pyogenes Cas9 protein (SpCas9), a nuclease inactive SpCas9 (dSpCas9), and a SpCas9 nickase (nSpCas9).
 312. The complex of claim 311, wherein the SaCas9 comprises the amino acid sequence provided in SEQ ID NO: 4273 or
 4274. 313. The complex of claim 312, wherein the SaCas9 comprises one or more of E781K, N967K, or R1014H mutations in the amino acid sequence provided in SEQ ID NO: 4273, or one or more corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 10-260 and
 4275. 314. The complex of claim 311, wherein the SpCas9 comprises the amino acid sequence provided in SEQ ID NO:
 4276. 315. The complex of claim 311, wherein the SpCas9 comprises a D9A mutation in the amino acid sequence provided in SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260.
 316. The complex of claim 303, wherein the Cas9 protein is derived from Corynebacterium ulcerous, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis I, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, or Neisseria meningitidis.
 317. The complex of claim 316, wherein the Cas9 protein is derived from Neisseria meningitidis.
 318. The complex of claim 303, wherein the cytidine deaminase: (i) is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase; (ii) comprises an amino acid sequence that is at least 85% identical to any of the amino acid sequences of SEQ ID NO: 266-284, 607-610, 5724-5736, and 5738-5741; (iii) comprises any of the amino acid sequences of SEQ ID NO: 266-284, 607-610, 5724-5736, and 5738-5741; (iv) is a rat APOBEC1 (rAPOBEC1) deaminase comprising one or more mutations selected from the group consisting of W90Y, R126E, and R132E of SEQ ID NO: 284, or one or more corresponding mutations in another APOBEC deaminase; (v) is a human APOBEC1 (hAPOBEC1) deaminase comprising one or more mutations selected from the group consisting of W90Y, Q126E, and R132E of SEQ ID NO: 5724, or one or more corresponding mutations in another APOBEC deaminase; (vi) is a human APOBEC3G (hAPOBEC3G) deaminase comprising one or more mutations selected from the group consisting of W285Y, R320E, and R326E of SEQ ID NO: 275, or one or more corresponding mutations in another APOBEC deaminase; (vii) is an activation-induced deaminase (AID); or (viii) is a cytidine deaminase 1 from Petromyzon marinus (pmCDA1).
 319. The complex of claim 303, wherein the cytidine deaminase is a cytidine deaminase from a human or a rat.
 320. The complex of claim 318, wherein the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase.
 321. The complex of claim 318, wherein the APOBEC family deaminase is an APOBEC3G deaminase.
 322. The complex of claim 303, wherein the UGI protein comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 322-324 and
 600. 323. The complex of claim 303, wherein the UGI protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 322-324 and
 600. 324. A method of nucleic acid editing, the method comprising contacting a target nucleic acid molecule with the complex of claim 303, wherein the method results in deamination of a cytidine in the target nucleic acid molecule.
 325. The method of claim 324, wherein the gRNA comprises a nucleotide sequence of at least 10 contiguous nucleotides that is complementary to the target nucleic acid molecule.
 326. The method of claim 324, wherein the target nucleic acid molecule comprises a sequence associated with a disease or disorder, and wherein the deamination of the cytidine results in a sequence that is not associated with the disease or disorder.
 327. The method of claim 324, wherein the target nucleic acid molecule comprises a point mutation associated with a disease or disorder, and wherein the deamination corrects the point mutation.
 328. The method of claim 327, wherein the target nucleic acid molecule comprises a T to C point mutation, and wherein the deamination of the mutant C base results in a nucleic acid sequence that is not associated with the disease or disorder.
 329. The method of claim 326, wherein the target nucleic acid sequence associated with the disease or disorder encodes a protein and wherein the deamination introduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein.
 330. A polynucleotide encoding two or more components of the complex of claim
 303. 331. A vector comprising the polynucleotide of claim
 330. 332. The vector of claim 331 further comprising a heterologous promoter that drives expression of the polynucleotide.
 333. A cell comprising the complex of claim
 303. 334. A cell comprising the polynucleotide of claim
 330. 335. A cell comprising the vector of claim
 331. 